Method of identifying diagnostic reagents

ABSTRACT

Disclosed herein are methods for identifying diagnostic reagents (e.g., antigen-binding molecules, such as antibodies) that are useful, for instance, as primary diagnostic, prognostic, and/or predictive (e.g., companion diagnostic) reagents in a disease state, such as cancer.

CROSS REFERENCE TO RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application Nos.60/949,792 filed Jul. 13, 2007 and 60/988,196 filed Nov. 15, 2007,herein incorporated by reference.

FIELD OF THE DISCLOSURE

This disclosure concerns a method for identifying diagnostic reagents(e.g., antigen-binding molecules, such as antibodies) that are useful,for instance, as primary diagnostic, prognostic, and/or predictive(e.g., companion diagnostic) reagents of a disease state, such ascancer.

BACKGROUND

Cancer is a generic name for a wide range of cellular malignanciescharacterized by unregulated growth, lack of differentiation, and theability to invade local tissues and metastasize. These neoplasticmalignancies affect, with various degrees of prevalence, every tissueand organ in the body. Historically, cancers have been diagnosed usingconventional histological and clinical features of the affected tissueor organ. However, it is now apparent that tumors, even from the sametissue or organ, are heterogeneous on the cellular and/or molecularlevel. As one consequence, the prognosis and/or responsiveness totherapy of each patient may differ. This unpredictability confoundstreatment selection and may expose patients to the risks and discomfortsof unneeded therapies or may lead to failure to treat a patient with abeneficial therapy.

EGFR-positive cancers offer a case in point. EGFR and its downstreamsignaling effectors, including members of the Ras/Raf/MAP kinasepathway, play an important role in both normal and malignant epithelialcell biology (Normanno et al., Gene, 366:2-16, 2006). Amplificationand/or mutation of the EGFR gene and/or EGFR protein overexpression havebeen associated with various malignancies, including breast cancer, lungcancer, colorectal cancer, ovarian cancer, renal cell cancer, bladdercancer, head and neck cancer, glioblastoma, and/or astrocytoma.Increased EGFR activity (whether as a result of abnormally high proteinexpression, dysregulation of receptor activity, or other mechanism) isbelieved to contribute to carcinogenesis. Consequently, EGFR is oneestablished target for therapeutic development.

Several EGFR inhibitors are available for clinical treatment. Theseinclude EGFR-specific antibodies (e.g., cetuximab (ERBITUX™) andpanitumumab (VECTIBIX™)) and small molecular tyrosine kinase inhibitors(e.g., gefitinib (IRESSA™) and erlotinib (TARCEVA™)). While thesetreatments have benefited subsets of cancer patients, responses to thedrugs are variable. For example, three clinical studies of patients withadvanced colorectal cancer using cetuximab in a monotherapy settingand/or in combination with irinotecan (a chemotherapeutic agent)demonstrated response rates of 10.5% or 10.8% for cetuximab alone and22.5% or 22.9% for the combined therapy (reviewed by Iqbal and Lenz,Cancer Chemother. Pharmacol., 54(Suppl. 1):532-39, 2004). Similarly,about 10% or about 20% of non-small cell lung cancer (“NSCLC”) patientstreated with 250 or 500 gefitinib per day, respectively, responded tothe drug and exhibited improved symptoms (Birnbaum and Ready, Curr.Treat. Options Oncol., 6(1):75-81, 2005).

Patient responses to EGFR inhibitors have been correlated with variousEGFR metrics. For example, EGFR expression (as measured byimmunohistochemistry) was associated with an objective response toerlotinib treatment in NSCLC patients (Tsao et al., N. Engl. J. Med.,353:133-144, 2005). However, survival after treatment in these patientswas not influenced by EGFR expression, the number of EGFR copies, orEGFR mutation (Tsao et al., N. Engl. J. Med., 353:133-144, 2005). Inboth preclinical and clinical settings, somatic mutations in the EGFRtyrosine kinase domain were found to correlate with sensitivity of NSCLCpatients to gefitinib and erlotinib but not to cetuximab (Janne et al.,J. Clin. Oncol., 23:3227-3234, 2005). Clinical studies of gefitinibdemonstrated an association between increased EGFR copy number,mutational status, and clinical response in advanced NSCLC (Cappuzzo etal., J. Natl. Cancer Inst., 97:643-655, 2005).

EGFR is only one of many cancer-related biomarkers that is used and/orconsidered for use as a prognostic marker and for which targeted cancertherapies have been and/or are being developed. Any cancer-relatedbiomarker that plays a meaningful role in cancer onset, growth (e.g.,proliferation and/or inhibition of apoptosis), metastasis,vascularization or the like is a candidate for a prognostic markerand/or for targeted therapy. Many of these biomarkers, the biologicalpathways in which they function, and the corresponding molecularpathogenesis(es) resulting in cancer are known. Now, a challenge is touse this information to identify useful diagnostic and therapeuticreagents to provide cancer patients with an accurate prediction ofdisease outcome and to administer “the appropriate drug, at theappropriate dose, at the appropriate time.”

SUMMARY OF THE DISCLOSURE

Disclosed herein are methods of identifying specific binding molecules(such as antibodies or fragments thereof) that interrogate the activitystate of a neoplasm (e.g., cancer)-related biomarker in biologicalsamples (such as formalin-fixed, paraffin-embedded (“FFPE”) tissuesections). In particular examples, the biomarker is a receptor tyrosinekinase (RTK), such as an oncogenic RTK. Activation status of suchbiomarker predicts, among other things, expression of the biomarker bythe tumor, the aggressiveness of the neoplasm comprising such biomarkerand/or the potential efficacy of therapies targeted to such biomarker.In particular examples the method of identifying a diagnostic reagentincludes identifying amino acid residues that form a protein-proteininterface between an RTK (e.g., a region of the intracellular domain ofRTK) and an RTK regulatory protein. A specific binding reagent isproduced that specifically binds at least some of the amino acidresidues that form the protein-protein interface, such as amino acids ofthe RTK intracellular domain that form a part of the interface, whereinthe binding occurs in the absence of the protein-protein interaction(e.g., the binding is detectable, for example above background) butfails to specifically bind amino acid residues that form theprotein-protein interface in the presence of the protein-proteininteraction (e.g., binding is not detectable, for example at abackground level).

Also provided are diagnostic reagents identified using these methods, aswell as methods of using such reagents to diagnose a subject withcancer, for example to estimate life expectancy (e.g., prognosis),determine the likely outcome of a particular anti-cancer therapy (e.g.,predictive), and to develop a treatment protocol for a subject withcancer.

The foregoing and other features will become more apparent from thefollowing detailed description of several embodiments, which proceedswith reference to the accompanying figures.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 is a flow chart schematically showing the steps of exemplaryimmunostaining methods.

FIG. 2 is a schematic representation of the EGFR regulatory domainshowing the locations of autophosphorylation sites (white boxes withnumber indicating position of autophosphorylated amino acid residue) andknown binding sites for exemplary EGFR regulatory proteins, includingSignal Transducer and Activator of Transcription (STAT) proteins (e.g.,STAT1 and/or STAT3; OMIM Accession Nos. 600555 or 102582), Growth FactorReceptor-Bound Protein 2 (Grb2; OMIM Accession No. 108355), Signalingand Transforming Protein Containing Src Homology 2 and 3 Domains (Shc)(e.g., OMIM Accession Nos. 600560, 605217, or 605263), Int-P, andSuppressor of Cytokine Signaling proteins (e.g., SOCS1 and/or SOCS3).

FIGS. 3A and 3B are a series of schematic representations involving anRTK molecule which, in its wild-type state, has an extracellularligand-binding domain (LBD) and intracellular tyrosine kinase (TKD) andregulatory (RD) domains. In each schematic, as applicable, EDBMrepresents an extracellular-domain-specific binding molecule (such as anantibody); RDBM represents a regulatory-domain-specific binding molecule(such as an antibody); and RDIM represents a regulatory domaininhibitory molecule (such as a SOCS protein like SOCS1 or SOCS3 or othernegative regulatory protein listed in Table 3) and RDSM represents aregulatory domain stimulatory molecule (such as a positive regulatoryprotein listed in Table 3). Each of the three schematics (from left toright) represents a different molecular setting. The left-most panelshows a full-length RTK not associated with a RDIM or RDSM. The centerpanel shows a constitutively active N-terminal truncated RTK mutant. Theright-most panel shows a full-length RTK associated with a RDIM or RDSM.In each case, predictions based on the binding (or not) of the EDBM andRDBM or RDSM are listed below the molecular schematic.

FIG. 4 shows the results of two Western blots in which three totalprotein concentrations (as indicated) of A431 (EGFR-positive) and BT474(EGFR-negative) cell lysates were run. The left and rightrepresentations show clone 5B7 and clone 3C6, respectively, binding to asingle protein band (appropriate in size for EGFR) in A431 cell lysates.Molecular weights (in kD) are shown at the far left.

FIG. 5 shows the results of staining the indicated tissues with EGFRextracellular-domain-specific clone 3C6 and EGFRregulatory-domain-specific clone 5B7 in tabular (FIG. 5A) and image(FIG. 5B) formats.

FIG. 6 shows images demonstrating the differences in the binding of EGFRregulatory-domain-specific (RDBM) clone 5B7 and EGFRexternal-domain-specific (EDBM) clone 3C6 in two representativenon-small cell lung cancer (NSCLC) tissue sections.

FIG. 7 shows the results of peptide inhibition studies mapping theepitope of the EGFR regulatory-domain-specific clone 5B7. The boxedregion in FIG. 7B represents an exemplary clone 5B7 epitope, theC-terminus of which may be a few amino acids longer or shorter.

FIG. 8 shows images demonstrating the differences in the binding of EGFRregulatory-domain-specific (RDBM) clone 5B7 and EGFRexternal-domain-specific (EDBM) clone 3C6 in two normal mouse livers(left-most two panels) and the livers of SOCS3-knock-out mice(right-most two panels).

FIG. 9 shows two Kaplan Meier plots demonstrating overall survival ofNSCLC patients as a function of clone 3C6 (panel A) or 5B7 (panel B)status. In panel A, 3C6-positive staining is shown by a black line and3C6-negative staining is shown by a gray line. In panel B, 5B7-positivestaining is shown by a black line and 5B7-negative staining is shown bya gray line.

FIG. 10 shows two Kaplan Meier plots demonstrating disease-free survivalof NSCLC patients as a function of clone 3C6 (panel A) or 5B7 (panel B)status. In panel A, 3C6-positive staining is shown by a black line and3C6-negative staining is shown by a gray line. In panel B, 5B7-positivestaining is shown by a black line and 5B7-negative staining is shown bya gray line.

SEQUENCE LISTING

The nucleic and amino acid sequences listed in the accompanying sequencelisting are shown using standard letter abbreviations for nucleotidebases, and three letter code for amino acids, as defined in 37 C.F.R.1.822. Only one strand of each nucleic acid sequence is shown, but thecomplementary strand is understood as included by any reference to thedisplayed strand. All sequence database accession numbers referencedherein are understood to refer to the version of the sequence identifiedby that accession number as it was available on the filing date of thisapplication. In the accompanying sequence listing:

SEQ ID NO: 1 is a reference amino acid sequence (REFSEQ) of human EGFR(isoform a) as set forth in GENBANK™ Accession No. NM_(—)005228. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)005228.

SEQ ID NO: 2 is the amino acid sequence of a peptide corresponding toresidues 1167-1185 of SEQ ID NO: 1.

SEQ ID NO: 3 is the curated reference amino acid sequence (REFSEQ) ofhuman Suppressor of Cytokine Signaling 3 (SOCS3) as set forth inGENBANK™Accession No. NM_(—)003955. A nucleic acid sequence encodingthis polypeptide also is set forth in GENBANK™ Accession No.NM_(—)003955.

SEQ ID NO: 4 is a reference amino acid sequence (REFSEQ) of human c-kit(isoform 1) as set forth in GENBANK™ Accession No. NM_(—)000222. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)000222.

SEQ ID NO: 5 is a reference amino acid sequence (REFSEQ) of human HER2(isoform a) as set forth in GENBANK™ Accession No. NM_(—)004448. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)004448.

SEQ ID NO: 6 is a reference amino acid sequence (REFSEQ) of human IGF1Ras set forth in GENBANK™ Accession No. NM_(—)000875. A nucleic acidsequence encoding this polypeptide also is set forth in GENBANK™Accession No. NM_(—)000875.

SEQ ID NO: 7 is a reference amino acid sequence (REFSEQ) of human c-Met(isoform b) as set forth in GENBANK™ Accession No. NM_(—)000245. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)000245.

SEQ ID NO: 8 is a reference amino acid sequence (REFSEQ) of human FGFR1(isoform 1) as set forth in GENBANK™ Accession No. NM_(—)023110. Anucleic acid sequence encoding this polypeptide also is set forth inGENBANK™ Accession No. NM_(—)023110.

DETAILED DESCRIPTION I. Introduction

The present disclosure provides methods for identifying one or morediagnostic reagents, such as a reagent that can be used to as adiagnostic (e.g., predictive, prognostic, or companion diagnostic) in adisease, for example a neoplasm, such as a tumor, for example cancer. Insome examples, the identified diagnostic reagent can be used as acompanion diagnostic to determine if a particular receptor tyrosinekinase (RTK) is present in a sample (e.g., to determine if a subject hasa neoplasm (e.g., cancer) expressing a particular RTK, such as HER2),determine the activity state of a cancer biomarker, for example theactivity state of a RTK (e.g., to determine the prognosis of a subjecthaving a neoplasm (e.g., cancer) or to predict the likelihood that asubject will respond to a particular therapy) or to identify treatmentsfor a subject with a neoplasm (e.g., cancer), or combinations thereof.

In particular examples the disclosed methods of identifying a diagnosticreagent include identifying an RTK that is a component of a cellularpathway that confers a growth advantage on a cancer cell (e.g., isoncogenic). In some examples, such an RTK is referred to herein as atarget RTK. When the RTK is inhibited in a cancer cell (e.g., thebiological activity or presence of the protein is substantiallydecreased or even eliminated), the cancer cell growth advantage isdecreased or eliminated (such as a decrease of at least 20%, at least40%, at least 50%, at least 75%, at least 90%, or even at least 99%). Asa result, for example, the cancer cell may undergo apoptosis or growthof the cancer cell may substantially slow, stop, or decrease. Incontrast, when the RTK is stimulated or activated in a cancer cell(e.g., the biological activity or presence of the protein issubstantially increased), the cancer cell growth advantage is increased(such as an increase of at least 20%, at least 40%, at least 50%, atleast 75%, at least 90%, or even at least 100%), thereby permitting oreven enhancing growth of the cancer cell.

RTKs useful for the disclosed methods are positively or negativelyregulated by a regulatory protein when the RTK and the regulatoryprotein specifically bind to one another to form a protein-proteininteraction. RTK-regulatory protein interactions are known in the art,and additional ones can be identified using routine methods (e.g.,immunoprecipitation, yeast two-hybrid system). The resultingprotein-protein interface results in the interaction between RTK aminoacids and regulatory protein amino acids. For example, at least 3, atleast 5, at least 10, at least 12, at least 15, at least 18, at least20, at least 25, at least 30 or even at least 50 amino acids (such as 3,4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22,23, 24, 25, 26, 27, 28, 29, 30, 35, 40, 45 or 50 amino acids) form aprotein-protein interface between an RTK and its regulatory protein. Inspecific examples, a portion or segment of the RTK intracellular domain(ID, such as a regulatory domain, RD) specifically binds to a regulatoryprotein, creating an interaction between amino acids of both proteins.

In particular examples, the method further includes identifying three ormore amino acid residues that form the protein-protein interface betweenthe RTK and its bound regulatory protein (e.g., a positive or negativeregulatory protein). Methods for identifying amino acids that comprise aprotein-protein interface are known in the art. In some examples, atleast 3, at least 5, at least 10, or at least 20 amino acid residues areidentified, such as 3 to 100, 5 to 100, 5 to 50, 5 to 30, 5 to 20, 10 to15, or 4 to 15 amino acids, for example 5 to 50 or 5 to 100 contiguousamino acids of the RTK or regulatory protein that forms the interface,such as 5 to 30, 5 to 25, 6 to 25, or 7 to 15 contiguous amino acids. Insome examples, at least some of such identified amino acids arecontiguous; however, one skilled in the art will appreciate that theinterface may not solely include contiguous amino acids.

A specific binding reagent can be generated to at least a portion of theamino acid residues that form the RTK protein-regulatory proteininterface using methods known in the art. In some examples, amino acidsfrom an RTK intracellular domain (ID), such as from the regulatorydomain (RD), are used to produce a specific binding reagent that candisrupt interaction between an RTK and its regulatory protein. In someexamples, amino acid residues that form the protein-protein interfacebetween the RTK and its bound regulatory protein are identified, and aspecific binding reagent generated to at least a portion of the aminoacid residues of the interface using methods known in the art. Such aninterface specific binding reagent can be used as a diagnostic (e.g.,companion diagnostic, prognostic, or predictive) reagent in thedisclosed methods. In some examples the specific binding reagent isspecific for amino acids of the RTK (such as the ID or RD of the RTK).The resulting specific binding reagent specifically binds (e.g.,detectable binding is observed or is significantly above backgroundlevels) at least one amino acid residue of the protein-protein interfacein the absence of the protein-protein interaction and fails tospecifically bind (e.g., detectable binding is not observed or is atbackground levels) at least one amino acid residue of theprotein-protein interface in the presence of the protein-proteininteraction.

One skilled in the art will appreciate that not all amino acid residuesof the protein-protein interface or of an RTK ID or RTK RD need beidentified or used to generate a specific binding reagent. In someexamples, only a portion of the amino acids forming the protein-proteininterface (or of the RTK RD or ID) are identified, such as at least 95%,at least 90%, at least 75%, at least 50%, or at least 25% of the aminoacids of the interface can be identified (such as 20 to 99%, for example50-95% of the amino acids). For example, if 20 amino acids form aprotein-protein interface between RTK and a regulatory protein, all 20amino acids need not be identified. Instead, for example, fewer than 20amino acids may be identified, such as 5 to 15 or 6 to 12 of such aminoacids. Similarly, not all identified amino acid residues of theprotein-protein interface or of an RTK ID need be used to generate aspecific binding reagent. As the regulatory protein need only stericallyhinder the binding of the specific binding agent to the protein-proteininterface (e.g., the RTK ID or RD), the epitope of the specific bindingagent may be entirely within the protein-protein interface or justpartially (e.g., as little as one amino acid) in the interface. So longas the specific binding agent does not significantly bind theprotein-protein interface when the regulatory protein is bound to theRTK, but does specifically bind the interface when the regulatoryprotein is not bound, it is sufficient. For example, an antigenic regionwithin the identified amino acid residues of the protein-proteininterface (e.g., an epitope) can be identified and used to generate aspecific binding reagent. For example, if 20 amino acids of theprotein-protein interface or an RTK RD are identified, all 20 aminoacids need not be used to generate a specific binding reagent, a portionthereof, such as at least 95%, at least 90%, at least 75%, at least 50%,or at least 25% of the identified amino acids can be used (such as 20 to99%, for example 50-95% of the amino acids) to generate a specificbinding agent.

Methods of producing specific binding reagents are known in the art. Inany embodiment involving a specific binding molecule (whethercomposition or method), a specific binding molecule can be (but is notnecessarily) an aptamer, an antibody (e.g., a monoclonal antibody, suchas a rabbit or mouse monoclonal antibody) or an antigen-binding fragmentthereof. In one example the method includes producing antibodies, suchas an antibody specific for amino acids of the RTK that form aprotein-protein interface with a regulatory protein. In particularexamples, the method includes producing antibodies specific for aminoacids of the RTK (e.g., amino acids of the ID or RD) that form all orpart of the protein-protein interface between the proteins. Some methodembodiments involve immunizing a non-human mammal with an immunogen thatincludes a carrier protein and amino acid residues that form theprotein-protein interface between RTK and its bound regulatory protein(or an immunogenic fragment of such an interface, such as an epitope ofthe interface). In some examples, an adjuvant is also administered tothe non-human mammal. Some such methods include a further step ofisolating serum from the non-human mammal and isolating polyclonalantibody specific for the immunogen. Other such methods include afurther step of fusing spleen cells from the non-human animals with afusion cell partner to make antibody-producing hybridomas.

Also disclosed are diagnostic reagents identified using the disclosedmethods, as well as compositions that include such reagents. Forexample, such detection reagents can be in a purified or isolated form.In some examples the identified reagents are present with other agentssuch as water, saline, or other liquid (e.g., buffered liquid) thatpermits suspension of the detection reagents. Such diagnostic reagentscan be present in a separate container, and part of a kit, such as a kitthat includes one or more other reagents for immunohistochemistry (IHC),such as labeled secondary antibodies (wherein the labeled antibodies areappropriately matched to the detection reagent, e.g., if the diagnosticreagent is a mouse monoclonal antibody, the secondary antibody is alabeled anti-mouse antibody), buffers, agents that permit development ordetection of the label, and the like.

Methods are provided for using the disclosed diagnostic reagents ascompanion diagnostics, for example to determine whether a samplecontains (e.g., is positive for) an RTK. For example, such methods canbe used to determine if a cancer expresses a particular RTK. Suchinformation can be useful in identifying treatment modalities for thesubject. For example, diagnostic reagents specific for theprotein-protein interface formed between an RTK intracellular domain(ID) and a regulatory protein can be used to determine if a samplecontaining cancer cells expresses a target RTK. In some examples, apositive-control specific binding reagent (e.g., an antibody specificfor an extracellular domain (ED) of the target RTK) is used incombination with the disclosed diagnostic reagents. As discussed above,diagnostic specific binding reagents identified using the disclosedmethods can bind to a protein-protein interface in the absence ofbinding of a regulatory protein to the RTK, but cannot bind to theinterface in the presence of binding of the regulatory protein to theRTK. Detection of specific binding of such a diagnostic reagent tocancer cells in a sample indicates that the cells express the RTK.Subject's having cancer cells that express the target RTK may benefitfrom an inhibitory RTK anti-cancer therapy (e.g., a tyrosine kinaseinhibitor (TKI)). In contrast, substantially no detectable binding ofthe specific binding agent to the cells indicates (but does not aloneconfirm) that the cells do not express the RTK. If substantially nospecific binding is detected, the cells in the sample may express thetarget RTK, but the epitope recognized by the specific binding reagentmay be masked by an interaction of the RTK with one or more regulatoryproteins with which the RTK forms protein-protein interaction(s). Insome method embodiments, it may be advantageous to confirm whether ornot the cells express the RTK by using a second diagnostic reagent thatrecognizes an ED of the target RTK (such as in the same tissue sectionor another tissue section in a series of sections). If both specificbinding agents are used, and substantially no specific binding isdetected using a disclosed diagnostic specific binding reagent (e.g.,which recognizes a target RTK ID), and substantially no specific bindingis detected using the specific binding reagent for the RTK ED, thisindicates that the cell does not express the target RTK. In contrast, ifsubstantially no specific binding is detected using the discloseddiagnostic reagent (e.g., one that recognizes the target RTK ID), andspecific binding is detected using the specific binding reagent for theRTK ED, this indicates the regulated status of the target RTK (e.g., thetarget RTK is down-regulated by a negative regulatory protein or thetarget RTK is up-regulated by a positive regulatory protein). In someembodiments, the regulated status of the target RTK further indicateswhether or not a subject may benefit from therapies that are intended toinhibit the target RTK. As such, the disclosed diagnostic reagents canbe used as prognostics, for example to predict the outcome of a subjecthaving cancer in the absence of administering additional therapy to thesubject. This is discussed in more detail below.

Disclosed methods also include using a diagnostic specific bindingreagent as a prognostic, for example to determine the prognosis of asubject by determining the activity state of an RTK, such as a subjecthaving a neoplasm (e.g., cancer). Thus, the disclosed diagnosticspecific binding reagents can be used to predict a subject's diseaseoutcome in the absence of administering additional therapies to thesubject. For example, such methods can be used to predict the prognosisof a neoplastic disease (such as a tumor or a cancer, for example lungcancer, colorectal cancer, head and neck cancer, gastric cancer, orglioblastoma). In some examples, the method includes detecting in abiological sample from a patient having a neoplastic disease thespecific binding of a disclosed diagnostic specific binding reagent thatis specific for the interface between an RTK and a negative regulator toone or more RTK-positive neoplastic cells in the biological sample;wherein the specific binding of the reagent in the one or moreRTK-positive neoplastic cells predicts a worse or poor prognosis of theneoplastic disease in the patient. In some such method embodiments, ifthe specific binding reagent (e.g., antibody or aptamer) specificallybinds to at least 10% (such as at least 15%, at least 20%, at least 30%,at least 50%, or at least 75%) of the RTK-positive neoplastic cells inthe biological sample, this indicates a positive result (e.g., positivefor binding). In contrast, in other examples the specific binding of adisclosed diagnostic specific binding reagent that is specific for theinterface between an RTK and a positive regulator to one or moreRTK-positive neoplastic cells in the biological sample is determined;wherein the absence of specific binding of the diagnostic specificbinding reagent in the one or more RTK-positive neoplastic cellspredicts a worse or poor prognosis of the neoplastic disease in thepatient. In some method embodiments, a poor prognosis is less than5-year survival (such as less than 1-year survival or less than 2-yearsurvival) of the patient after initial diagnosis of the neoplasticdisease.

Other prognostic method embodiments involve detecting in a biologicalsample from a patient having a neoplastic disease (such as lung cancer,colorectal cancer, head and neck cancer, gastric cancer, orglioblastoma) the specific binding of a disclosed diagnostic specificbinding reagent that is specific for the interface between an RTK and anegative regulator to one or more RTK-positive neoplastic cells in thebiological sample is determined; wherein substantially no specificbinding of the diagnostic specific binding reagent in the one or moreRTK-positive neoplastic cells predicts a good prognosis of theneoplastic disease in the patient. For example, this result indicatesthat the subject has a cancer in which a target RTK that is “naturally”inhibited by its protein-protein interaction with a negative regulatoryprotein, and is thus likely to have a better outcome than a cancer inwhich such negative regulation of the oncogenic target RTK is lacking(and vice versa). The presence of binding of a negative regulator to thetarget RTK indicates that the RTK is marked for degradation as aconsequence of its protein-protein interaction with a negativeregulatory protein (e.g., in the ubiquitin-dependent proteasome pathway,in a ubiquitin-independent proteasome pathways, or in a 20S or 26Sproteasome-dependent pathway) and thus cannot confer a continuing growthadvantage on a cancer cell. As a result, prognosis for a subject withsuch a cancer is likely to be better than a patient with an oppositeresult (i.e., oncogenic target RTK not marked for degradation). Incontrast, in other examples the specific binding of a discloseddiagnostic specific binding reagent that is specific for the interfacebetween an RTK and a positive regulator to one or more RTK-positiveneoplastic cells in the biological sample is determined; whereinspecific binding of the diagnostic specific binding reagent in the oneor more RTK-positive neoplastic cells predicts a good prognosis of theneoplastic disease in the patient. In some method embodiments, a goodprognosis is greater than 2-year survival (such as greater than 3-yearsurvival, greater than 5-year survival, or greater than 7-year survival)of the patient after initial diagnosis of the neoplastic disease.

In addition to using the disclosed diagnostic specific binding reagentsto determine the aggressiveness of a neoplasm (e.g., prognosis of theneoplasm), the disclosed diagnostic specific binding reagents can alsobe used to predict the response of a neoplasm to RTK inhibitor therapy.Thus, the disclosed diagnostic specific binding reagents can be used aspredictors of the likelihood that a particular tumor will respond to anRTK inhibitor therapy. In some examples, the methods include detectingin a biological sample, which includes one or more neoplastic cells(such as cancer cells), the specific binding of a disclosed diagnosticspecific binding reagent that is specific for the protein-proteininterface between an RTK and a negative regulator (e.g., a bindingreagent specific for an RTK ID) to the one or more of the neoplasticcells; wherein specific binding of the specific binding reagent to oneor more of the neoplastic cells indicates that the neoplastic cells willrespond to an inhibitor of the RTK. For example, the detection ofbinding of a disclosed specific binding reagent to a sample indicatesthat a negative regulatory protein is not bound to the RTK, and thus theRTK is “available” for binding to an administered RTK inhibitor (e.g.,an inhibitor that recognizes an RTK ID, such as a TKI). In some methodembodiments, the specific binding of the specific binding reagent to atleast 10% (such as at least 15%, at least 20%, at least 30%, at least50%, or at least 75%) of the neoplastic cells in the biological sampleindicates that the candidate is likely to respond to treatment with anRTK inhibitor. In some method embodiments, the neoplastic cell responseto the RTK inhibitor is slowed growth (such as, net zero growth or netnegative growth). In other method embodiments, the slowed growth is atleast 10% (such as at least 15%, at least 20%, at least 30%, at least50%, or at least 75%) less than the neoplastic cell growth prior totreatment with the RTK inhibitor. In some method embodiments, theneoplastic cell response to the RTK inhibitor is apoptosis, and, in somesuch embodiments, at least 10% (such as at least 15%, at least 20%, atleast 30%, at least 50%, or at least 75%) of the neoplastic cellsundergo apoptosis.

Other disclosed methods involve predicting the response of a neoplasm toan RTK inhibitor by detecting in a biological sample comprising one ormore RTK-positive neoplastic cells substantially no specific binding ofa disclosed specific binding reagent that is specific for theprotein-protein interface between an RTK and a negative regulator (e.g.,a RTK ID-binding antibody) to the one or more RTK-positive neoplasticcells; wherein substantially no specific binding of the specific bindingreagent to the RTK-positive neoplastic cells indicates that theneoplastic cells will not substantially respond to an RTK inhibitor thatis specific for the RTK ID. Without wishing to be bound to a particulartheory, it is proposed that the absence of such binding indicates thatthe RTK is being “naturally” inhibited (by the negative regulator towhich it is bound) and thus the RTK inhibitory therapy has less (ornothing) left to inhibit. Thus, in the absence of any therapy the targetRTK is unlikely to be further inhibited by a therapy designed to inhibitthe target RTK; and a patient receiving such RTK inhibitory therapy isunlikely to respond to it. In a more particular example, an target RTKthat has been marked for degradation as a consequence of itsprotein-protein interaction with a negative regulatory protein (e.g., inthe ubiquitin-dependent proteasome pathway, in a ubiquitin-independentproteasome pathways, or in a 20S or 26S proteasome-dependent pathway) isunlikely to be further inhibited by a therapy designed to inhibit thetarget RTK; thus, again, a patient receiving such therapy is unlikely torespond to it. Some such methods further involve detecting in a controlbiological material (such as normal skin, normal testis, or normaltonsil) the specific binding to RTK of the specific binding reagent.Other such methods further involve detecting in the biological samplespecific binding of a second specific binding reagent (e.g., antibody)specific for the RTK external domain (ED).

In some examples, the methods include detecting in a biological sample,which includes one or more neoplastic cells (such as cancer cells), thespecific binding of a disclosed diagnostic specific binding reagent thatis specific for the protein-protein interface between an RTK and apositive regulator (e.g., a binding reagent specific for an RTK ID) tothe one or more of the neoplastic cells; wherein the absence of specificbinding of the specific binding reagent to one or more of the neoplasticcells indicates that the neoplastic cells will respond to an inhibitorof the RTK. Without wishing to be bound to a particular theory, it isproposed that the absence of binding of a disclosed specific bindingreagent to a sample indicates that the oncogenic RTK is very active, andthus there is a significant amount of RTK activity to be inhibited. Insome method embodiments, the neoplastic cell response to the RTKinhibitor is slowed growth (such as, net zero growth or net negativegrowth). In other method embodiments, the slowed growth is at least 10%(such as at least 15%, at least 20%, at least 30%, at least 50%, or atleast 75%) less than the neoplastic cell growth prior to treatment withthe RTK inhibitor. In some method embodiments, the neoplastic cellresponse to the RTK inhibitor is apoptosis, and, in some suchembodiments, at least 10% (such as at least 15%, at least 20%, at least30%, at least 50%, or at least 75%) of the neoplastic cells undergoapoptosis.

Other disclosed methods involve predicting the response of a neoplasm toan RTK inhibitor by detecting in a biological sample comprising one ormore RTK-positive neoplastic cells significant specific binding of adisclosed specific binding reagent that is specific for theprotein-protein interface between an RTK and a positive regulator (e.g.,a RTK ID-binding antibody) to the one or more RTK-positive neoplasticcells; wherein substantial specific binding of the specific bindingreagent to the RTK-positive neoplastic cells indicates that theneoplastic cells will not substantially respond to an RTK inhibitor thatis specific for the RTK ID. In some method embodiments, the specificbinding of the specific binding reagent to at least 10% (such as atleast 15%, at least 20%, at least 30%, at least 50%, or at least 75%) ofthe neoplastic cells in the biological sample indicates that thecandidate is not likely to respond to treatment with an RTK inhibitor.Without wishing to be bound to a particular theory, it is proposed thatthe presence of such binding indicates that the oncogenic RTK is lessactivity than if the positive regulator were present, and thus the RTKinhibitory therapy has less to inhibit. Some such methods furtherinvolve detecting in a control biological material (such as normal skin,normal testis, or normal tonsil) the specific binding to RTK of thespecific binding reagent. Other such methods further involve detectingin the biological sample specific binding of a second specific bindingreagent (e.g., antibody) specific for the RTK external domain (ED).

Still other disclosed methods involve predicting the response of aneoplasm to RTK inhibitor (e.g., a TKI) administration, by detecting RTKexpression in a first sample of a biological material comprising one ormore neoplastic cells; and detecting in a second sample of thebiological material substantially no specific binding to RTK of adisclosed specific binding reagent; wherein detecting RTK expression inthe first sample and substantially no specific binding to RTK of adisclosed specific binding reagent indicates that the neoplasm is likelyto respond to RTK inhibitor (e.g., a TKI) administration. In some suchmethods, the first sample and the second sample are serial sections ofthe biological material. Other such methods further involve detecting ina control biological material (such as, normal skin, normal testis, ornormal tonsil) the specific binding to RTK of the disclosed specificbinding reagent (e.g., RKT ID-binding antibody).

Immunostaining methods also are disclosed. Such methods can involvecontacting a biological sample containing one or more cells with adisclosed specific binding reagent (e.g., RKT ID-binding antibody oraptamer), and detecting the specific binding of the specific bindingreagent to an antigen (e.g., an RTK ID) in the one or more cells. Insome examples, a labeled secondary antibody is used to detect thespecific binding reagent (e.g., RKT ID-binding antibody). However, oneskilled in the art will appreciate that other routine immunostainingmethods can be used.

Other disclosed methods involve detecting a direct interaction betweenan RTK and an RTK regulatory protein (such as a positive or negativeregulatory protein), by contacting a biological sample, comprising oneor more RTK-positive cells, with a disclosed specific binding reagent(e.g., RKT ID-binding antibody), and detecting the specific binding ofthe specific binding reagent to the one or more RTK-positive cells,wherein the specific binding of the specific binding reagent to the oneor more RTK-positive cells detects that the RTK is not significantlyinteracting with an RTK regulatory protein, wherein an interactionbetween RTK and the RTK regulatory protein masks the epitope of thespecific binding reagent (e.g., RKT ID-binding antibody).

Other methods of detecting a direct interaction between an RTK and anRTK regulatory protein (such as a positive or negative regulatoryprotein) are disclosed. Such methods can include contacting a biologicalsample that includes one or more RTK-positive cells, with a disclosedspecific binding reagent (e.g., RKT ID-binding antibody), and detectingthe specific binding of the specific binding reagent to the one or moreRTK-positive cells, wherein substantially no specific binding of thespecific binding reagent to the one or more RTK-positive cells detectsthat the RTK is interacting with an RTK regulatory protein, wherein aninteraction between RTK and the RTK regulatory protein masks the epitopeof the specific binding reagent (e.g., RKT ID-binding antibody).

In any disclosed method embodiment involving a biological sample, suchbiological sample can be (but is not necessarily) mounted on amicroscope slide, is a tissue section (such as a formalin-fixed andparaffin-embedded tissue section), and/or is a neoplastic tissue (suchas a cancer, for example a lung cancer, colorectal cancer, head and neckcancer, gastric cancer, or glioblastoma).

Provided herein is a particular example of identifying a diagnosticspecific binding agent for EGFR. Using the disclosed methods, anantibody specific for the EGFR regulatory domain (EGFR-RD) wasidentified. It was confirmed that this antibody can be used to diagnoseand prognose a subject having an EGFR-expressing neoplasm. One skilledin the art will appreciate that similar methods can be used for otherRTKs, and for other RTK-regulatory binding proteins, using the methodsprovided herein.

Disclosed herein are EGFR regulatory domain peptides (“RDPs”), whichinclude, e.g., isolated peptides consisting of amino acid residues1167-1185 of SEQ ID NO: 1 or an immunogenic fragment of said peptide.Also disclosed are (EGFR) regulatory domain (RD)-binding molecules thatspecifically binds to such peptides. Some embodiments include aRD-binding molecule that specifically binds to residues 1138-1196 of SEQID NO: 1 or a SOCS-protein-binding fragment thereof (e.g., aSOCS3-binding fragment). Also disclosed are compositions including anEGFR RD-binding molecule the binding of which to EGFR is competitivelyinhibited by a disclosed RDP (such as an isolated peptides consisting ofamino acid residues 1167-1185 of SEQ ID NO: 1 or an immunogenic fragmentof said peptide).

II. Abbreviations and Terms

ED extracellular (or external) domain of RTK EGFR epidermal growthfactor receptor (e.g., OMIM Accession No. 131550) FFPE formalin-fixed,paraffin-embedded FGFR fibroblast growth factor receptor (e.g., OMIMAccession No. 136350 for FGFR1, 176943 for FGFR2, 134934 for FGFR3, and134935 for FGFR4) HER human epidermal growth factor receptor (e.g., OMIMAccession No. 164870 for HER2 (ErbB2), 190151 for HER3 (ErbB3), and600543 for HER4 (ErbB4)) HGFR (Met) hepatocyte growth factor receptor(e.g., OMIM Accession No. 164860) ID intracellular (or internal) domainof RTK IGFR insulin-like growth factor receptor (e.g., OMIM AccessionNo. 147370 for IGF1R and 147280 for IGF2R) IHC ImmunohistochemistryPDGFR Platelet derived growth factor receptor (e.g., OMIM Accession No.173490 and 173410) NSCLC non-small cell lung cancer RDP regulatorydomain peptide RD regulatory domain of RTK RET rearranged followingtransfection (e.g., OMIM Accession No. 164761) RON recepteur d'originenantais receptor (e.g., OMIM receptor Accession No. 600168) RTK receptortyrosine kinase SH2 domain Src homology 2 domain SOCS suppressor ofcytokine signaling protein (e.g., OMIM protein Accession Nos. 604176 or603597) STAT signal transducer and activator of transcription VEGFvascular endothelial growth factor receptor (e.g., OMIM Accession No.165070 for VEGFR1, 191306 for VEGFR2, and 136352 for VEGFR3)

Unless otherwise noted, technical terms are used according toconventional usage. Definitions of common terms in cell and molecularbiology may be found in Benjamin Lewin, Genes V, published by OxfordUniversity Press, 1994 (ISBN 0-19-854287-9); Kendrew et al. (eds.), TheEncyclopedia of Molecular Biology, published by Blackwell Science Ltd.,1994 (ISBN 0-632-02182-9); and Robert A. Meyers (ed.), Molecular Biologyand Biotechnology: a Comprehensive Desk Reference, published by VCHPublishers, Inc., 1995 (ISBN 1-56081-569-8).

In order to facilitate review of various embodiments of a disclosedinvention, the following explanations of specific terms are provided:

Antigen-binding molecule: A molecule that specifically binds to anepitope in a target molecule (e.g., an antigen, such as a protein ornucleic acid molecule). Exemplary antigen-binding molecules are providedelsewhere in this disclosure, and include, for example, antibodies andaptamers.

Species of antigen-binding molecules described herein include, withoutlimitation, interface-specific binding molecules, RTK-binding molecules,RTK ID-binding molecules, RTK RD-binding molecules, regulatoryprotein-binding molecules, and control antigen-binding molecules. Thesespecies of antigen-binding molecules are characterized by the nature ofthe target molecule and/or the location in the target molecule of theepitope to which the species specifically binds as more particularlydefined elsewhere in this disclosure. In some examples, the targetmolecule is an intracellular domain of RTK that specifically binds to aregulatory protein.

Cancer: Malignant neoplasm, for example one that has undergonecharacteristic anaplasia with loss of differentiation, increased rate ofgrowth, invasion of surrounding tissue, and is capable of metastasis.

Contact: To bring one agent into close proximity to another agent,thereby permitting the agents to interact. For example, an antibody (orother specific binding agent) can be applied to a microscope slide orother surface containing a biological sample, thereby permittingdetection of proteins (or protein-protein interactions orprotein-nucleic acid interactions) in the sample that are specific forthe antibody.

Detect: To determine if an agent or interaction (e.g., binding betweentwo proteins or a protein and a nucleic acid) is present or absent. Insome examples this can further include quantification. For example, useof an antibody specific for a particular protein (e.g., an RTK) or aparticular protein-protein interface, permits detection of the of theprotein or protein-protein interaction in a sample, such as a samplecontaining cancer tissue. In particular examples, an emission signalfrom a label is detected. Detection can be in bulk, so that amacroscopic number of molecules can be observed simultaneously.Detection can also include identification of signals from singlemolecules using microscopy and such techniques as total internalreflection to reduce background noise.

Diagnose: The process of identifying a medical condition or disease, forexample from the results of one or more diagnostic procedures.Diagnostic reagents can be used as companion diagnostics, asprognostics, and as predictors. In a specific example, a neoplasm, suchas a cancer, is diagnosed in a subject by detecting the activationstatus of an RTK protein associated with the neoplasm.

In some examples, diagnosis includes determining whether or not asubject has a particular disease, such as whether the subject has acancer that expresses a particular RTK. In particular examples,diagnosis includes determining the prognosis of a subject, such asdetermining the likely outcome of a subject having a disease in theabsence of additional therapy (e.g., life expectancy). In yet otherexamples, diagnosis includes predicting a subject's response to aparticular therapy, such as predicting the likely outcome of treating asubject's tumor with an RTK inhibitor.

Epidermal growth factor receptor (EGFR): The cell-surface receptor formembers of the epidermal growth factor family of extracellular proteinligands. EGFR is a member of the ErbB family of receptors, a subfamilyof four closely related receptor tyrosine kinases: EGFR (ErbB-1),HER2/c-neu (ErbB-2), Her 3 (ErbB-3) and Her 4 (ErbB-4). Mutationsaffecting EGFR expression or activity can result in cancer, and thusEGFR can have oncogenic activity. Exemplary sequences are providedherein.

Epitope: A site on a target molecule (e.g., an antigen, such as aprotein or nucleic acid molecule) to which an antigen-binding molecule(e.g., an antibody, antibody fragment, scaffold protein containingantibody binding regions, or aptamer) binds. Epitopes can be formed bothfrom contiguous or juxtaposed noncontiguous residues (e.g., amino acidsor nucleotides) of the target molecule (e.g., a protein-proteininterface). Epitopes formed from contiguous residues (e.g., amino acidsor nucleotides) typically are retained on exposure to denaturingsolvents whereas epitopes formed by tertiary folding typically are loston treatment with denaturing solvents. An epitope typically includes atleast 3, and more usually, at least 5 or 8-10 residues (e.g., aminoacids or nucleotides). Typically, an epitope also is less than 20residues (e.g., amino acids or nucleotides) in length, such as less than15 residues or less than 12 residues.

Human epidermal growth factor receptor 2 (HER2): A member of the ErbBprotein family, which is a proto-oncogene located at the long arm ofhuman chromosome 17(17q11.2-q12). Approximately 25-30% of breast cancershave an amplification of the HER2/neu gene or overexpression of itsprotein product, referred to as “HER2 positive” (HER2+). HER2+ patientsmay receive the monoclonal antibody trastuzumab (Herceptin), which bindsthe HER2ED, as a therapy for breast cancer. Overexpression of HER2 inbreast cancer has been associated with increased disease recurrence andworse prognosis.

Immunogen: A molecule (also called an antigen) capable of provoking animmune response (e.g., the production of antibodies) when introducedinto an animal with a functioning immune system. Exemplary immunogensincluding, for instance, proteins (or protein fragments) such as an RTKor portion thereof, polysaccharides, and small molecules (haptens) orpeptides coupled to a carrier molecule (e.g., a protein such as bovineserum albumin (“BSA”), keyhole limpet hemocyanin (“KLH”) or polylysine).An “immunogenic fragment” is a portion of a polypeptide or otherimmunogen that is capable of provoking an immune response either byitself or when conjugated to a carrier molecule. Immunogens andimmunogenic fragments include one or more epitopes within theirsequences.

Isolated: An “isolated” biological component (e.g., a nucleic acidmolecule, chemical compound, protein or organelle) has beensubstantially separated or purified away from other biologicalcomponents (e.g., nucleic acid molecules, chemical compounds, proteinsor organelles) with which the component is comingled (e.g., in the cellof an organism or in a plant cell extract). Nucleic acids, proteins andchemical compounds that have been “isolated” include nucleic acids,proteins and chemical compounds purified by standard purificationmethods. The term “isolated” also embraces nucleic acids and proteinsprepared by recombinant expression in a host cell as well as chemicallysynthesized nucleic acids and chemical compounds.

Label: An agent capable of detection, for example by spectrophotometry,flow cytometry, or microscopy. For example, one or more labels can beattached to an antibody, thereby permitting detection of a targetprotein or a target protein-protein interaction. Exemplary labelsinclude radioactive isotopes, fluorophores, ligands, chemiluminescentagents, enzymes, and combinations thereof.

Neoplasm: Abnormal growth of cells.

Normal cells or tissue: Non-tumor, non-malignant cells and tissue.

Peptide: Two or more amino acids joined by a peptide bond. Typically, apeptide consists of fewer than fifty amino acids; for example,consisting of approximately 7 to approximately 40 amino acids,consisting of approximately 7 to approximately 30 amino acids,consisting of approximately 7 to approximately 20 amino acids.

Sample: A biological specimen, such as one that includes detectableproteins and/or nucleic acids, obtained from a subject. Examplesinclude, but are not limited to, peripheral blood, urine, saliva, tissuebiopsy, surgical specimen, bone marrow, amniocentesis samples andautopsy material. In one example, a sample includes proteins. In someexamples, the sample is a tissue sample obtained from a subject known tohave, or suspected to have, cancer. Samples, such as tissue samples, canbe placed on microscope slides. In particular examples, samples are useddirectly, or can be manipulated prior to use, for example, by fixing(e.g., using formalin) or embedding (e.g., in plastic or paraffin).

Specific binding (or obvious derivations of such phrase, such asspecifically binds, specific for, etc.) refers to the particularinteraction between one binding partner (such as an RTK, for example anID of the RTK) and another binding partner (such as a regulatory proteinof the RTK, for example a positive or negative regulator, or an antibodyor aptamer specific for an RTK protein-regulatory protein interface).Such interaction is mediated by one or, typically, more noncovalentbonds between the binding partners (or, often, between a specific regionor portion of each binding partner). In contrast to non-specific bindingsites, specific binding sites are saturable. Accordingly, one exemplaryway to characterize specific binding is by a specific binding curve. Aspecific binding curve shows, for example, the amount of one bindingpartner (the first binding partner) bound to a fixed amount of the otherbinding partner as a function of the first binding partnerconcentration. As the first binding partner concentration increasesunder these conditions, the amount of the first binding partner boundwill saturate. In another contrast to non-specific binding sites,specific binding partners involved in a direct association with eachother (e.g., a protein-protein interaction) can be competitively removed(or displaced) from such association (e.g., protein complex) by excessamounts of either specific binding partner. Such competition assays (ordisplacement assays) are very well known in the art.

Subject: Includes any multi-cellular vertebrate organism, such as humanand non-human mammals (e.g., veterinary subjects). In some examples, asubject is one who has cancer, or is suspected of having cancer. In someexamples veterinary subjects are used to produce antibodies, such as inmice, rabbits, cows, or chickens.

Tumor: A neoplasm. Includes solid and hematological (or liquid) tumors.

Unless otherwise explained, all technical and scientific terms usedherein have the same meaning as commonly understood by one of ordinaryskill in the art to which a disclosed invention belongs. The singularterms “a,” “an,” and “the” include plural referents unless contextclearly indicates otherwise. Similarly, the word “or” is intended toinclude “and” unless the context clearly indicates otherwise.“Comprising” means “including.” Hence “comprising A or B” means“including A” or “including B” or “including A and B.

Suitable methods and materials for the practice and/or testing ofembodiments of the disclosure are described below. Such methods andmaterials are illustrative only and are not intended to be limiting.Other methods and materials similar or equivalent to those describedherein can be used. For example, conventional methods well known in theart to which a disclosure pertains are described in various general andmore specific references, including, for example, Sambrook et al.,Molecular Cloning: A Laboratory Manual, 2d ed., Cold Spring HarborLaboratory Press, 1989; Sambrook et al., Molecular Cloning: A LaboratoryManual, 3d ed., Cold Spring Harbor Press, 2001; Ausubel et al., CurrentProtocols in Molecular Biology, Greene Publishing Associates, 1992 (andSupplements to 2000); Ausubel et al., Short Protocols in MolecularBiology: A Compendium of Methods from Current Protocols in MolecularBiology, 4th ed., Wiley & Sons, 1999; Harlow and Lane, Antibodies: ALaboratory Manual, Cold Spring Harbor Laboratory Press, 1990; and Harlowand Lane, Using Antibodies: A Laboratory Manual, Cold Spring HarborLaboratory Press, 1999.

III. Receptor Tyrosine Kinases

Receptor tyrosine kinases (RTKs) are cell surface receptor proteins thatinclude an extracellular ligand-binding domain (ED) (e.g., capable ofbinding a growth factor, cytokine, or hormone), a transmembrane spanningdomain, and an intracellular domain (ID) responsible for kinaseactivity. Most RTKs are single subunit receptors but some (e.g., theinsulin receptor) are multimeric complexes. Ligand binding to the EDinduces RTK dimerization and autophosphorylation. The resultingactivated phosphorylated RTK can then function to recruit intracellularsignaling proteins via its ID (such as a Src homology 2 (SH2) domain).Thus, ligand binding to the ED generates a signal inside the cell viathe ID. For example, phosphorylation of specific tyrosine residueswithin the activated RTK creates binding sites for SH2- andphosphotyrosine binding (PTB) domain-containing regulatory proteins.Binding of regulatory proteins to the RTK ID initiates signaltransduction pathways. Other regulatory proteins that interact with theactivated RTK may function as adaptor proteins and have no intrinsicenzymatic activity of their own, but can link RTK activation todownstream signal transduction pathways, such as the MAP kinasesignaling cascade.

There are several RTK families, wherein each generally has relatedligands and similar extracellular domains. Exemplary families useful forthe methods provided herein include those shown in Table 1.

TABLE 1 Exemplary RTK families Family Exemplary members Platelet derivedgrowth factor colony stimulating factor 1 (CSF-1), receptor (PDGFR)PDGFRA, PDGFRB, c-Kit Fibroblast growth factor receptor FGFR1, FGFR2,FGFR3, FGFR4 (FGFR) Insulin receptor insulin receptor, insulin likegrowth factor-1 (IGF1R), IGF2R Epidermal growth factor receptor EGFR,HER2 (ErbB2), HER3 (EGFR) (ErbB3), HER4 (ErbB4) Vascular endothelialgrowth factor VEGFR 1 (Flt1) VEGFR 2 receptor (VEGFR) (KDR/Flk-1),VEGFR3 (Flt4), Flt3 Trk receptors trkA, trkB and trkC Hepatocyte growthfactor receptor c-Met, RON (HGFR) RET Ret EPH EphA1-8, 10; EphB1-4, 6UFO/Axl/Tyro-3 Axl, Mer, Tyro3

RTKs that can be used in the methods provided herein include those thatare oncogenic, that is, that can cause or induce the development oftumors, such as cancer. Generally, RTK oncogenes can cause unregulatedcell growth of a cancer cell, for example by activating signalingproteins involved in cellular proliferation as well as activatingphosphoinositide 3-kinase (PI3-K)-dependent pathways which can alsocontribute to oncogenesis (for example by producing an anti-apoptoticsignal). For example, oncogenic RTKs can confer a growth advantage to acancer cell in which the RTK is expressed, such as when a positiveregulatory protein is specifically bound to the RTK ID. For example, apositive RTK regulator may increase RTK biological activity (such asdownstream effects) or expression, or prevent or reduce RTK degradation(or combinations thereof). In contrast, inhibition of an oncogenic RTKcan reduce or eliminate the growth advantage to a cancer cell, such aswhen a negative regulatory protein is specifically bound to the RTK ID.For example, a negative RTK regulator may target the RTK fordegradation, decrease biological activity (such as downstream effects)or decrease RTK expression (or combinations thereof). Therefore, use ofthe diagnostic reagents obtained using the methods herein can be used todetermine the activation status of a target RTK, and thus allow one topredict or determine the aggressiveness of a neoplasm (e.g., cancer)expressing the RTK, the potential efficacy of therapies targeted to theRTK, and determine if a subject has a neoplasm that expresses the RTK.

In some examples, an oncogenic RTK is one whose amino acid sequencediffers from the native RTK sequence (e.g., RET, Kit), resulting in aprotein that is not regulated in the same manner as the native RTKsequence (e.g., unregulated cell proliferation). In some examples, anoncogenic RTK has the same sequences as a native or wild-type sequence(e.g., EGFR, MET) but which becomes significantly upregulated (e.g., dueto the binding of a positive regulator or loss of binding of a negativeregulator).

Exemplary RTKs that can be used in the methods provided herein include,but are not limited to, those listed in Table 2. RTK protein sequencesare publicly available, for example on GenBank or EMBL websites.Exemplary non-limiting examples include those shown in Table 2. Oneskilled in the art will appreciate that other sequences can be used inthe methods provided herein, such as those from other mammals besideshumans, as well as polymorphic and mutant variants thereof

TABLE 2 Exemplary GenBank Accession Numbers for RTK Protein SequencesRTK GenBank Accession Nos.* EGFR NM_005228; NM_201282; NM_201283;NM_201284 FGFR1 NM_023105; NM_023106; NM_023110; NM_023111; NM_015850FGFR2 NM_000141; NM_022970 FGFR3 NM_000142; NM_022965 FGFR4 NM_213647;NM_002011; NM_022963 Flt3 NM_004119 HER2 (ErbB2) NM_004448; NM_001005862HER3 (ErbB3) NM_001982; NM_001005915 HER4 (ErbB4) NM_005235;NM_001042599 HGFR (Met) NM_000245; NM_001127500 IGF1R NM_000875 KitNM_000222; NM_001093772 PDGFR (PDGFRA NM_006206; NM_002609 and PDGFRB)Ret NM_020975; NM_020630 RON (MST1R) NM_002447 VEGFR1 (Flt1) NM_002019VEGFR2 (KDR/flk-1) NM_002253 VEGFR3 (Flt4) NM_182925; NM_002020*sequences associated with GenBank Accession Numbers as of Jul. 11, 2008are herein incorporated by reference.

As described above, activated RTKs can form specific protein-proteininteractions with positive and negative regulatory proteins. Exemplarypositive and negative regulatory proteins for particular RTKs are listedin Table 3. For example, the ID of an oncogenic RTK can bind with highspecificity to a regulatory protein. Such an interaction can form aprotein-protein interface, resulting in positive or negative regulationof the RTK. For example binding of a positive regulatory protein to theRTK ID can increase the biological activity of the RTK, increase thebiological activity of downstream activators, decrease degradation ofRTK, or combinations thereof. Binding of a positive regulator to RTK cantherefore confer a growth advantage to a cancer cell expressing the RTK.In contrast, binding of a negative regulatory protein to the RTK ID candecrease the biological activity of the RTK, decrease the biologicalactivity of downstream activators, increase degradation of RTK, orcombinations thereof. Binding of a negative regulator to RTK cantherefore decrease or eliminate a growth advantage to a cancer cellexpressing the RTK.

TABLE 3 Exemplary Positive and Negative Regulators of RTKs RTK PositiveRegulators Negative Regulators EGFR STAT1, STAT5B, GRB7, SOCS1, SOCS3,HER2, MUC1 SOCS5, CBL, LRIG1, SHP1, PKC FGFR1 GRB2, GRB4, GRB14, SOS1,SEF SHC1 FGFR2 CBL, SEF FGFR3 GRB2, STAT1, STAT3, SHP2 CBL FGFR4 STAT1,STAT3 Flt3 GRB2, SHC1 SOCS1 HER2 GRB2, GRB7, SOS1, SHC1, CAV1, LRIG1,CHIP (ErbB2) SHP2 HER3 GRB2, GRB7, SHC1 CBL, LRIG1 (ErbB3) HER4 GRB2,STAT5, SHC1, SHP2 CBL, LRIG1, PKC (ErbB4) HGFR (Met) GRB2, SHC1, STAT3,SRC, CBL, PKC, LRIG SHP2 IGF1R IRS1-4, GRB10, SHC1 SOCS1, SOCS2, SOCS3Kit GRB2, GRB7, GRB10, SHP1, SOCS1, STAT1, STAT5, SHP2, SOCS6, CBL, PKCPDGFR GRB2, GRB10, GRB14, CAV1, CAV3, CBL (PDGFRA SHP2, SHC1 and PDGFRB)Ret IRS1, GRB2, GRB7, GRB10, DOK4 SOS1, SHC, SHANK3, SH2B1 RON GRB2,SHC1, SRC CBL VEGFR1 GRB2, STAT1, STAT3, CBL (Flt1) SHC1, SHP2 VEGFR2GRB2, GRB10, SHC1, SHC2, SHP1, CBL, PKC (KDR/flk-1) STAT1, SHP2 VEGFR3GRB2, SHC1, STAT3, SRC, CBL (Flt4) SHP2

Although particular regulatory proteins are provided in Table 3, oneskilled in the art will appreciate that others are publicly known. Inaddition, additional regulatory proteins can be identified using routinemethods. For example, regulatory proteins that interact with an RTK (orportion thereof, such as an ID or RD thereof) can be identified usingimmunoprecipitation, a two-hybrid system (e.g., by using the RTK ID orRD as the “bait”), or pull-down assays. For example, an RTK of interestcan be expressed in a cell, and isolated from the cell using an antibodyto the RTK (e.g., an antibody that recognizes an ED or non RD of theRTK). In another example, an RTK of interest can be expressed in a cellalong with a marker, thereby resulting in expression of an RTK-fusionprotein (such as a 6X-HIS-RTK protein) which can be isolated from thecell using a molecule specific for the marker (e.g., Ni beads). Otherproteins that are isolated with the RTK complex (e.g., regulatoryproteins) can be then identified, for example by using western blottingor mass spectrometry methods.

Using the methods provided herein, a diagnostic binding reagent can beidentified or generated that specifically binds to at least a portion ofthe RTK protein-regulatory protein interface in the absence of the RTKprotein-regulatory protein interaction but does not specifically bindthe protein-protein interface in the presence of the RTKprotein-regulatory protein interaction. Positive and negative RTKregulatory proteins are known (e.g., see Table 3). In such examples, aparticular RTK/regulatory protein combination may be selected based onthe desired diagnostic reagent desired. For example, if diagnosis of aHER2-expressing tumor is desired, for example to determine the prognosisof a subject having a HER2-expressing tumor, a diagnostic bindingreagent that specifically binds to at least a portion of the amino acidsthat form a HER2-negative regulator protein-protein interface (e.g., aninterface between HER2 and one of CAV1, CBL or LRIG1) or a HER2-positiveregulator protein-protein interface (e.g., an interface between HER2 andone of GRB2, GRB7, SOS1, SHP2, or SHC1) but does not specifically bindthe protein-protein interface in the presence of the protein-proteininteraction, can be generated. However, one skilled in the art willappreciate that a diagnostic binding reagent can be identified withoutknowing the identity of the regulatory protein. For example, all thatmay be known is that the regulatory protein is a negative or positiveregulator. In specific examples, amino acids (e.g., at least 5) thatmake up a least a portion of a protein-protein interface between an RTKand a regulatory protein listed in Table 3 are identified and used toproduce a diagnostic specific binding agent.

RTKs associated with oncogenesis are known in the art. Some RTKs, suchas EGFR, are associated with many different cancer types. However, someRTKs are associated with particular cancers, such as those listed inTable 4. When generating a diagnostic specific binding agent using themethods provided herein, an RTK can be identified or selected based onthe neoplasm or cancer for which a diagnostic specific binding agent isdesired. For example, if a general cancer diagnostic reagent is desired,an RTK that is oncogenic for several cancers can be selected, such asEGFR or HGFR. If a diagnostic reagent for a particular cancer type isdesired, an RTK associated with the cancer can be selected, for exampleas shown in Table 4. For example, if a diagnostic reagent for prostatecancers that express VEGFR1 is desired, the VEGFR1RTK can be selected asthe RTK for which a diagnostic reagent can be generated.

TABLE 4 Exemplary cancers associated with RTKs RTK Cancer EGFR non-smallcell lung, bladder, cervical, ovarian, kidney and pancreatic cancer,squamous head and neck carcinoma, glioblastoma multiforme FGFR1Glioblastoma FGFR2 Breast cancer FGFR3 Bladder cancer, multiple myelomaFGFR4 Breast cancer, prostate cancer, rhabdomyosarcoma Flt3 Acutemyeloid leukemia, acute lymphoblastic leukemia HER2 breast cancer, coloncancer, ovarian cancer, non-small cell (ErbB2) lung cancer HER3Non-small cell lung cancer, breast cancer (ErbB3) HER4 Prostate cancer,breast cancer, colorectal cancer (ErbB4) HGFR Thyroid papillarycarcinoma, multiple papillary renal cell (MET) carcinoma, colorectalcancer, breast cancer, non-small cell lung cancer, ovarian cancer,pancreatic cancer IGF1R gastrointestinal neuroendocrine tumor, breastcancer, prostate cancer Kit gastrointestinal stromal tumors, mast celldisease, acute myelogenous leukemia, melanoma, thyroid carcinoma PDGFRgastrointestinal stromal tumor, glioblastoma Ret Neuroblastoma,medullary thyroid carcinoma, multiple endocrine neoplasias type II andIII, phaeochromocytoma and parathyroid tumors RON breast cancer, coloncancer, pancreatic cancer VEGFR1 non-small cell lung cancer, colorectalcancer, breast cancer, (Flt1) bladder cancer, prostate cancer VEGFR2breast cancer, prostate cancer, ovarian cancer (KDR/ flk-1) VEGFR3breast cancer, non-small cell lung cancer (Flt4)

IV. Identifying Amino Acids that Form a Protein-Protein Interface

This disclosure provides, among other things, methods for producing adiagnostic binding reagent that specifically binds to at least a portionof the RTK protein-regulatory protein interface in the absence of theRTK protein-regulatory protein interaction but does not specificallybind the protein-protein interface in the presence of the RTKprotein-regulatory protein interaction. In some examples, prior togenerating such a diagnostic specific binding reagent, at least aportion of the amino acids that form the RTK protein-regulatory proteininterface are identified. For example, if diagnosis of a VEGF-expressingtumor is desired, for example to determine the prognosis of a subjecthaving a VEGF-expressing tumor, at least three amino acids that form aVEGF-negative regulator protein-protein interface (e.g., an interfacebetween VEGF1 and one of GRB2, STAT1, STAT3, SHC1 or SHP2) or aVEGF1-positive regulator protein-protein interface (e.g., an interfacebetween VEGF1 and CBL) can be identified, if not already known in theart. In some examples, only amino acids of the RTK ID or RD that arepart of the interface are identified (e.g., regulatory protein aminoacids are not identified).

Methods are provided for identifying protein-protein interactions, forexample in biological samples (e.g., isolated cells or tissues). Suchmethods can be used to identify amino acids that form a protein-proteininterface between an RTK ID and a regulatory protein to permitproduction of diagnostic reagents for use in determining the activitystate of an RTK. Upon specific binding of a regulatory protein to the IDof an RTK, certain amino acids of the RTK ID will interact with certainamino acids of the regulatory protein. By identifying at least a subsetof the amino acids that form the resulting protein-protein interface,diagnostic specific binding reagents can be generated that can be usedto determine the activity state of an RTK.

Amino acid residues that form an interface between an RTK and one of itsregulatory proteins (such as a positive or negative regulator, e.g., seeTable 3) are identified. Although not all amino acids that form theinterface need be identified, a number of amino acids is identified thatpermits generation of a diagnostic specific binding reagent thatspecifically binds to at least a subset (e.g., at least one) of theamino acid residues of the protein-protein interface in the absence ofthe RTK protein-regulatory protein interaction and fails to specificallybind amino acid residues of the RTK protein-regulatory protein interfacein the presence of the RTK protein-regulatory protein interaction. Oneskilled in the art will appreciate that the specific binding reagentneed not bind all amino acids that comprise the protein-proteininterface. So long as the specific binding reagent binds to theinterface in the absence of regulatory protein binding to the RTK, anddoes not bind the interface when regulatory protein is bound to the RTK,it can be used in the disclosed methods. Thus, in some examples thespecific binding reagent need only bind at least 1 amino acid of theinterface, such as at least 2, for example 1 to 5, 1 to 10 or 5 to 20amino acids of the interface. In particular examples, a sufficientnumber of amino acids are identified that permit generation of aspecific binding reagent, such as identification of an immunogenic orantigenic peptide. For example, immunogenic peptides can produce asignificant antibody response in an animal (e.g., rabbit, mouse, orchicken) immunized with the peptide under routine conditions for formingantibodies.

In particular examples, amino acids of the RTK ID that form part of theprotein-protein interface are identified, such amino acids of theregulatory domain that form part of the protein-protein interface. Forexample, at least 3, at least 5, at least 10, or at least 20 amino acidsof the protein-protein interface can be identified, such as 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 40, 50, or 100amino acids, for example 5 to 8, 5 to 12, 8 to 10, 8 to 12, 8 to 15, or8 to 20 amino acids. In one example, at least 3, at least 5, at least10, or at least 20 amino acids of an RTK ID (or RD) that forms aprotein-protein interface with a regulatory protein is identified, suchas 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, or 100 amino acids of an RTK ID (or RD), for example 5 to 8, 5to 12, 8 to 10, 8 to 12, 8 to 15, or 8 to 20 amino acids of an RTK ID(or RD).

One skilled in the art will recognize that a protein-protein interfaceformed by binding of a RTK ID to a regulatory protein may includenon-consecutive amino acid residues from each protein. That is, althougha protein-protein interface may span numerous amino acids (e.g., mayspan 100 amino acids of an RTK), not all amino acids in the span mayform or be part of the protein-protein interface (e.g., maybe only 5 to20 of the 100 RTK amino acids are part of the protein-proteininterface). Therefore, the amino acids identified and used to generate aspecific binding reagent may or may not be contiguous, or may include acombination of contiguous and non-contiguous amino acids. For example,amino acids 20-22, 30, 32 and 40-50 of an RTK ID may bind to amino acids5-10 and 30-40 of a regulatory protein. In such an example, at leastthree or at least five of such amino acids, such as amino acids 20-22and 40-43 of the RTK ID, can be identified.

In some examples, a positive or a negative regulatory protein isselected. That is, if a diagnostic reagent for determining whether anRTK is bound to a positive regulator, amino acids that form an interfacebetween such two proteins can be identified, while if a diagnosticreagent for determining whether an RTK is bound to a negative regulator,amino acids that form an interface between such two proteins can beidentified. Table 3 above provides exemplary known positive and negativeregulators for particular RTKs. Thus for example, if a diagnosticreagent for determining if Kit is bound to one of its negativeregulators (e.g., SHP1, SHP2, SOCS1, SOCS6, CBL) or for determining ifRet is bound to the positive regulator IRS1, GRB2, GRB7, GRB10, SOS1, orSHC, then amino acids forming at least a portion of the protein-proteininterface between such proteins can be identified.

In some examples, the identity of the regulator is not known prior toidentifying amino acids of the protein-protein interface (e.g., notknown whether it is a positive or negative regulator, or it is knownthat it is a positive or negative regulatory protein, but the specificidentity of the protein is not known). Once the specific binding agentis generated, it can subsequently determined if the bound regulator is apositive or negative regulatory protein (e.g., by determining the effectof the regulator on cancer cell growth, wherein a positive regulatoryprotein is one that enhances such growth while a negative regulatoryprotein is one that decreases such growth).

Biological components (e.g., proteins) that form direct interactions(such as protein-protein interactions) are known to those of ordinaryskill in the art. Various exemplary protein-protein interactions,including RTK-regulatory protein interactions, can be identified asshown in Table 3, as well as in one or more of the following publiclyavailable databases: AllFuse (European Bioinformatics Institute),Alanine Scanning Energetics DataBase (ASEdb; Harvard University),Binding Interface Database (BID; A & M University Texas); The GeneralRepository for Interaction Datasets (BioGRID; Samuel Lunenfeld ResearchInstitute); Biomolecular Object Network Databank (BOND; Thomson Corp.);Database of Interacting Proteins (DIP; UCLA); Genomic Knowledge Database(RIKEN, Institute of Physical and Chemical Research); HIV-1/HumanProtein Interaction Database (NCBI); Human Protein Intercation Database(HPID; Inha University); Human Protein Reference Database (Johns HopkinsUniversity and The Institute of Bioinformatics, India); Inter-ChainBeta-Sheets database (ICBS; University of California); Kinetic Data ofBio-molecular Interactions (KDBI; National University of Singapore);Biomolecular Relations in Information Transmission and Expression (KEGGBRITE; Kyoto University); Molecular INTeractions database (MINT; CBM,Rome); Mammalian Protein-Protein Interaction database (MPPI; MIPS);PDZBase (Weill Medical College of Cornell University); POINT (NationalHealth Research Institutes & National Taiwan University); PRoteinInteractions and Molecular Information databasE (PRIME; Human GenomeCenter, University of Tokyo); Protein Interaction Database (ProteinLounge); SNAPPIView (University of Dundee).

Some of the foregoing databases further identify the residues or regionsof the applicable proteins involved in the protein-protein interface.Therefore, in some examples the amino acids of the protein-proteininterface are known in the art. Alternatively, residues or regionsinvolved in a protein-protein interaction can be determined using anytechnique known to the ordinarily skilled artisan; for example, peptidecompetition studies (where a peptide having a sequence corresponding toresidues believed to be involved in a protein-protein interface is usedto competitively inhibit the protein-protein interaction; successfulinhibition by the peptide of the interaction indicates that the subjectsequence likely is involved in the protein-protein interaction),mutational analysis of one or both components of the protein-proteininteraction, or crystallography of the RTK-regulatory protein (or aportion thereof; for example an RTK ID) complex.

Methods of identifying amino acids that form a protein-protein interfacebetween two proteins are routine. In some examples an activated RTK isincubated with a regulatory protein in solution (e.g., using purifiedproteins) under conditions that permit the two proteins to bind and forma protein-protein interface. In other examples, a cell, such as a cancercell, that expresses the RTK and the regulatory protein is underconditions that permit the two proteins to bind and form aprotein-protein interaction, resulting in a protein-protein interface.The resulting protein-protein interaction can then be analyzed, forexample identifying at least three or at least five amino acids thatform the protein-protein interface (such as identifying RTC ID aminoacids that form a protein interface with the regulatory protein).

A. Competition Studies

In one example, peptide competition studies are performed to identifyamino acid residues that form a protein-protein interface between an RTKand a regulatory protein. Such methods are routine in the art. Forexample, peptide fragments of a target RTK ID (e.g., RTK RD, see Table5) can be generated. Such fragments can include contiguous amino acidsof the RTK ID, but can also include non-contiguous amino acids.Exemplary peptide fragments are at least 3 amino acids in length, suchas at least 5, at least 10, or at least 20 amino acids in length, suchas 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,40, 50, or 100 amino acids in length, for example 5 to 8, 5 to 12, 8 to10, 8 to 12, 8 to 15, or 8 to 20 amino acids in length, such as thisnumber of amino acids of an RTK RD. Peptide fragments can be generatedrandomly (e.g., by forming a library of molecules that span an entireRTK ID) or can be generated to specific sequence(s) corresponding toresidues believed to be involved in a protein-protein interface betweenan RTK and a regulatory protein. In some examples, peptide fragments aregenerated (for example using a computer program) that are suspected ofnot only of being involved in a protein-protein interface, but are alsosuspected to be immunogenic.

The resulting protein fragments are incubated with a regulatory proteinand an RTK protein (or portions thereof) to competitively inhibit theprotein-protein interaction that would normally result between the RTKand the regulatory protein. For example, purified regulatory and RTKproteins (or portions thereof) can be incubated in vitro or in vivo (forexample the fragment can be incubated with or introduced into a cancercell that expresses the activated RTK and regulatory proteins) withdifferent concentrations of the fragment peptide (e.g., serialdilutions, for example in the range of 0.01 μM to 10,000 μM, 0.01 μM to10,000 μM, or 1 μM to 1000 μM) and effects on binding between the RTKand its regulatory protein determined using routine protein detectionmethods, such as Western blotting, immunoprecipitation, andimmunostaining. If the peptide fragment significantly reduces orinhibits the protein-protein interaction, this indicates that thesubject sequence of the fragment likely is involved in theprotein-protein interaction. Such identified sequences can be used togenerate diagnostic specific binding reagents. Similarly, functionalstudies (e.g., cancer cell growth) can be performed in the presence andabsence of the competing peptides, to identify peptides that interferewith the binding of a regulatory peptide to an RTK. For example,identification of peptides that target an RTK-positive regulator can beselected by identifying those peptides that decrease the growth ofcancer cells in culture relative to the absence of the peptide (e.g., adecrease of at least 20%, such as at least 50%, or at least 80%)(decreased cancer cell growth in the presence of the peptide indicatesthat the positive regulator is not bound to the RTK, and thus the growthadvantage provided by the RTK is reduced). Alternatively, identificationof peptides that target a RTK-negative regulator can be selected byidentifying those peptides that increase the growth of cancer cells inculture relative to the absence of the peptide (e.g., an increase of atleast 20%, such as at least 50%, or at least 80%) (increased growth inthe presence of the peptide indicates that the negative regulator is notbound to the RTK, and thus the growth advantage provided by the RTK isincreased, as the RTK is no longer targeted for degradation).

B. Mutagenesis Studies

In another example, mutagenesis studies are performed to identify aminoacid residues that form a protein-protein interface between an RTK and aregulatory protein. Such methods are routine in the art. For example,deletion mutants of either the target RTK ID (e.g., RTK RD, see Table 5)or deletion mutants of the regulatory protein can be generated (e.g.,using recombinant molecular biology methods). In one example, aplurality of deletion mutants of a target RTK ID (or RD) are generated,and can include deletion of contiguous or non-contiguous amino acids (orcombinations thereof). Exemplary deletions include deletion of at least3 amino acids, such as at least 5, at least 10, or at least 20 aminoacids, such as 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19,20, 25, 30, 40, 50, or 100 amino acids, for example 5 to 8, 5 to 12, 8to 10, 8 to 12, 8 to 15, or 8 to 20 amino acids, such as this number ofamino acids deleted from an RTK RD or regulatory protein. MutagenizedRTK or regulatory proteins can be generated randomly (e.g., by forming alibrary of deletion mutants hat span an entire RTK ID or RD) or can begenerated to specific sequence(s) corresponding to residues believed tobe involved in a protein-protein interface between an RTK and aregulatory protein.

The resulting mutant proteins (e.g., mutant RTK+mutant regulatoryprotein; mutant RTK+native regulatory protein, or native RTK+mutantregulatory protein) are incubated under conditions that would normallypermit the native RTK protein and native regulatory protein to interact.For example, purified regulatory and RTK proteins (e.g., mutantRTK+mutant regulatory protein; mutant RTK+native regulatory protein, ornative RTK+mutant regulatory protein) can be incubated in vitro or invivo (for example the proteins can be incubated with or introduced intoa cancer cell that expresses the proteins) and effects on bindingbetween the RTK and its regulatory protein determined using routineprotein detection methods, such as Western blotting,immunoprecipitation, and immunostaining For example, if a mutant RTK isunable to bind to its native regulatory protein (and thus the mutationsignificantly reduces or inhibits the protein-protein interaction), thisindicates that the subject deleted sequence is likely involved in theprotein-protein interaction. The sequence of the deletion can be used togenerate diagnostic specific binding reagents.

Similarly, functional studies (e.g., cancer cell growth) can beperformed with various combinations of the native and mutant peptides(e.g., mutant RTK+mutant regulatory protein; mutant RTK+nativeregulatory protein, or native RTK+mutant regulatory protein), toidentify mutants that do not permit binding between a regulatory peptideand an RTK. For example, identification of deletion mutants that targetan RTK-positive regulator can be selected by identifying those deletionmutants that decrease the growth of cancer cells (e.g., a decrease of atleast 20%, such as at least 50%, or at least 80%) (decreased cancer cellgrowth in the presence of the deletion mutant indicates that thepositive regulator is not bound to the RTK, and thus the growthadvantage provided by the RTK is reduced). Alternatively, identificationof deletion mutants that target an RTK-negative regulator can beselected by identifying those deletion mutants that increase the growthof cancer cells (e.g., an increase of at least 20%, such as at least50%, or at least 80%) (increased cancer cell growth in the presence ofthe deletion mutant indicates that the negative regulator is not boundto the RTK, and thus the growth advantage provided by the RTK isincreased, as the RTK is no longer targeted for degradation). Furtherrefinements to an identified mutant sequence that results in decreasedbinding between an RTK and a regulatory protein can be made by sitedirected mutagenesis. For example if a region of 20 amino acids isidentified as playing a role in the protein-protein interaction, each ofthe 20 amino acids can be mutated individually or in variouscombinations and tested as described above, to permit identification ofthe particular residues that comprise the protein-protein interface.Such identified residues can be used to generate a specific bindingagent.

C. Crystallography

In another example, crystallography is used to identify amino acids ofan RTK protein-regulatory protein interface. For example, purified RTKand regulatory protein (or fragments thereof, such as an RTK ID or RD)can be incubated under appropriate conditions to allow the proteins tobind and crystallize, and an X-ray diffraction pattern obtained and usedto build an electron density map using tools well known to those skilledin the art of crystallography and X-ray diffraction techniques. For anoverview of the procedures of collecting, analyzing, and utilizing X-raydiffraction data for the construction of electron densities see, forexample, Campbell et al., Biological Spectroscopy, The Benjamin/CummingsPublishing Co., Inc., Menlo Park, Calif., 1984; Cantor et al.,Biophysical Chemistry, Part II: Techniques for the study of biologicalstructure and function, W.H. Freeman and Co., San Francisco, Calif.1980; A. T. Brunger, X-plor Version 3.1: A system for X-raycrystallography and NMR, Yale Univ. Pr., New Haven, Conn. 1993; M. M.Woolfson, An Introduction to X-ray Crystallography, Cambridge Univ. Pr.,Cambridge, UK, 1997; J. Drenth, Principles of Protein X-rayCrystallography (Springer Advanced Texts in Chemistry), Springer Verlag;Berlin, 1999; and Tsirelson et al., Electron Density and Bonding inCrystals: Principles, Theory and X-ray Diffraction Experiments in SolidState Physics and Chemistry, Inst. of Physics Pub., 1996. Information onmolecular modeling can be found for example in, M. Schlecht, MolecularModeling on the PC, 1998, John Wiley & Sons; Gans et al., FundamentalPrincipals of Molecular Modeling, Plenum Pub. Corp., 1996; N. C. Cohen(editor), Guidebook on Molecular Modeling in Drug Design, AcademicPress, 1996; and W. B. Smith, Introduction to Theoretical OrganicChemistry and Molecular Modeling, 1996.

Typically, a well-ordered crystal that will diffract x-rays strongly isused to solve the three-dimensional structure of a RTK protein bound toa regulatory protein by x-ray crystallography. The crystallographicmethod directs a beam of x-rays onto a regular, repeating array of manyidentical molecules. The x-rays are diffracted from it in a pattern fromwhich the atomic positions of the atoms that make up the protein-proteininterface of interest can be determined.

Substantially pure and homogeneous protein samples are usually used forcrystallization. Typically, crystals form when molecules areprecipitated very slowly from supersaturated solutions. A typicalprocedure for making protein crystals is the hanging-drop method, inwhich a drop of protein solution (e.g., that includes RTK protein,regulatory protein, and RTK protein bound to regulatory protein) isbrought very gradually to supersaturation by loss of water from thedroplet to the larger reservoir that contains salt, polyethylene glycol,or other solution that functions as a hydroattractant, although anyother method that generates diffraction quality crystals can be used. Insome examples diffraction quality crystals are obtained by seeding thesupersaturated solution with smaller crystals that serve as templates.

Powerful x-ray beams can be produced from synchrotron storage ringswhere electrons (or positrons) travel close to the speed of light. Theseparticles emit very strong radiation at all wavelengths from short gammarays to visible light. When used as an x-ray source, only radiationwithin a window of suitable wavelengths is channeled from the storagering.

In diffraction experiments a narrow and parallel beam of x-rays is takenout from the x-ray source and directed onto the crystal to producediffracted beams. The incident x-ray beam causes damage to both proteinand solvent molecules. The crystal is, therefore, usually cooled toprolong its lifetime (for example to −220° to −50° C.). In someexamples, single crystals are used to obtain a data set, while in otherexamples; multiple crystals are used to obtain a data set. The x-raybeam must strike the crystal from many different directions to produceall possible diffraction spots, thereby creating a complete data set.Therefore, the crystal is rotated relative to the beam during datacollection. The diffracted spots are recorded either on a film, or by anelectronic detector, both of which are commercially available.

When the primary beam from an x-ray source strikes the crystal, x-raysinteract with the electrons on each atom in the crystal and cause themto oscillate. The oscillating electrons serve as a new source of x-rays,which are emitted in almost all directions in a process referred to asscattering. When atoms (and hence their electrons) are arranged in aregular three-dimensional array, as in a crystal, the x-rays emittedfrom the oscillating electrons interfere with one another. In mostcases, these x-rays, colliding from different directions, cancel eachother out; those from certain directions, however, will add together toproduce diffracted beams of radiation that can be recorded as a patternon a photographic plate or detector.

The diffraction pattern obtained in an x-ray experiment is related tothe crystal that caused the diffraction. X-rays that are reflected fromadjacent planes travel different distances, and diffraction only occurswhen the difference in distance is equal to the wavelength of the x-raybeam. This distance is dependent on the reflection angle, which is equalto the angle between the primary beam and the planes.

Each atom in a crystal scatters x-rays in all directions, and only thosethat positively interfere with one another, according to Bragg's law (2dsin θ=λ), give rise to diffracted beams that can be recorded as adistinct diffraction spot above background. Each diffraction spot is theresult of interference of all x-rays with the same diffraction angleemerging from all atoms. To extract information about individual atomsfrom such a system requires considerable computation. The mathematicaltool that is used to handle such problems is called the Fouriertransform.

Each diffracted beam, which is recorded as a spot on the film, isdefined by three properties: the amplitude, which is measured as theintensity of the spot; the wavelength, which is determined by the x-raysource; and the phase information, which is lost in x-ray experimentsand must be calculated. All three properties are used for all of thediffracted beams, in order to determine the position of the atoms givingrise to the diffracted beams. Methods of determining the phases are wellknow in the art. For example, phase differences between diffracted spotscan be determined from intensity changes following heavy atomderivatization. Another example would be determining the phases bymolecular replacement.

The amplitudes and the phases of the diffraction data from the proteincrystals are used to calculate an electron-density map of the repeatingunit of the crystal. A model of the particular amino acid sequence isbuilt to approximate the electron density map. Such information can beused to identify amino acids that form a protein-protein interface thatresults when an RTK protein binds to a regulatory protein.

The initial model may contain some errors. Provided the protein crystalsdiffract to high enough resolution (e.g., better than 3.5 Å), most orsubstantially all of the errors can be removed by crystallographicrefinement of the model using computer algorithms. In this process, themodel is changed to minimize the difference between the experimentallyobserved diffraction amplitudes and those calculated for a hypotheticalcrystal containing the model. This difference is expressed as an Rfactor (residual disagreement) which is 0.0 for exact agreement andabout 0.59 for total disagreement.

Typically, the R factor of a refined model is between 0.15 and 0.35(such as less than about 0.24-0.28) for a well-determined proteinstructure. The residual difference is a consequence of errors andimperfections in the data. These derive from various sources, includingslight variations in the conformation of the protein molecules, as wellas inaccurate corrections both for the presence of solvent and fordifferences in the orientation of the microcrystals from which thecrystal is built. Thus, the final model represents an average ofmolecules that are slightly different, both in conformation andorientation.

In refined structures at high resolution, there are usually no majorerrors in the orientation of individual residues, and the estimatederrors in atomic positions are usually around 0.1-0.2 Å, provided theamino acid sequence is known.

Most x-ray structures are determined to a resolution between 1.7 Å. and3.5 Å. Electron-density maps with this resolution range are preferablyinterpreted by fitting the known amino acid sequences into regions ofelectron density in which individual atoms are not resolved.

Upon identification of amino acids that form the protein-proteininterface, diagnostic specific binding reagents can be generated thatrecognize at least a portion of this sequence.

V. RTK Intracellular Domains

In some examples, as an alternative to (or in addition to) identifyingamino acids of the protein-protein interface formed upon binding of aregulatory protein to an RTK to generate diagnostic specific bindingreagents, amino acids of the RTK ID, such as the RTK RD, are used togenerate diagnostic specific binding reagents in accordance with themethods provided herein. In some examples such RTK ID or RD amino acidsare identified and used to generate diagnostic specific bindingreagents, or if already known (e.g., see Table 5), can be simply used togenerate diagnostic specific binding reagents.

Table 5 provides exemplary known RTK IDs as well as portions thereof,such as RDs and for some inhibitory domains. For example, the RTK EGFRhas an EGFR regulatory domain (corresponding to residues 980-1210 of SEQID NO: 1), which includes an inhibitory subdomain (corresponds toresidues 1138-1196 of SEQ ID NO: 1). With this information, specificbinding reagents can be generated that specifically bind to anRTK-regulatory protein interface (for example by binding to the RTK ID,RD, or inhibitory domains) in the absence of RTK-regulatory proteinbinding, but fail to specifically bind to an RTK-regulatory proteininterface (e.g., fail to bind the RTK ID, RD or inhibitory domains) inthe presence of RTK-regulatory protein binding. For example, using themethods provided in Section VI below and the information in Table 5,specific binding reagents can be generated to the RTK ID, RD orinhibitory domain listed in Table 5 (e.g., by identifying epitopeswithin or that include these regions), and screened using routinemethods. In some examples, the information in Table 5 is used toidentify a protein-protein interface as described in Section IV above.For example, peptide fragments of the RTK ID and RD regions or RTKproteins with mutations in the ID or RD regions can be generated andtested as described above to identify amino acids comprising theprotein-protein interface.

TABLE 5 Exemplary RTK intracellular domains Intracellular RegulatoryInhibitory Tyrosine RTK domain domain domain Kinase domain EGFR aa669-1210 of aa 980-1210 of aa 1138-1196 aa 712-968 of SEQ ID NO 1 SEQ IDNO 1 of SEQ ID NO 1 SEQ ID NO 1 (NM_005228) (NM_005228) (NM_005228)(NM_005228) FGFR1 aa 398-822 of aa 755-822 of aa 478-754 of NM_023110NM_023110 NM_023110 FGFR2 aa 399-822 of aa 759-822 of aa 482-758 ofNM_022970 NM_022970 NM_022970 FGFR3 aa 396-806 of aa 749-806 of aa472-748 of NM_000142 NM_000142 NM_000142 FGFR4 aa 389-798 of aa 744-798of aa 467-743 of NM_002011 NM_002011 NM_002011 Flt3 aa 565-993 of aa944-993 of aa 610-943 of NM_004119 NM_004119 NM_004119 HER2 aa 676-1255of aa 977-1255 of aa 720-976 of NM_004448 NM_004448 NM_004448 HER3 aa667-1342 of aa 966-1342 of aa 709-965 of NM_001982 NM_001982 NM_001982HER4 aa 676-1292 of aa 719-1292 of aa 718-974 of NM_001042599NM_001042599 NM_001042599 HGFR aa 956-1390 of aa 1338-1390 of aa1078-1337 of (MET) NM_000245 NM_000245 NM_000245 IGF-1R aa 956-1367 ofaa 1267-1367 of aa 1275-1367 aa 999-1266 of NM_000875 NM_000875 ofNM_000875 NM_000875 Kit aa 544-976 of aa 925-976 of aa 589-924 ofNM_000222 NM_000222 NM_000222 PDGFRB aa 557-1106 of aa 959-1106 of aa600-958 of NM_002609 NM_002609 NM_002609 Ret aa 658-1114 of aa 1006-1114of aa 724-1005 of NM_020975 NM_020975 NM_020975 RON aa 983-1400 of aa1342-1400 of aa 1082-1341 of NM_002447 NM_002447 NM_002447 VEGFR1 aa780-1338 of aa 1155-1338 of aa 827-1154 of (Flt1) NM_002019 NM_002019NM_002019 VEGFR2 aa 766-1356 of aa 1161-1356 of aa 1161-1199 aa 834-1160of (KDR/flk- NM_002253 NM_002253 of NM_002253 1) NM_002253 VEGFR3 aa799-1363 of aa 846-1363 of aa 845-1169 of (Flt4) NM_182925 NM_182925NM_182925

For example, if a specific binding agent for assessing the activitystate of Ret was desired, amino acids 658-1114 or 1006-1114 of GenBankAccession No. NM_(—)020975 could be used to generate antibodies oraptamers, for example by identifying one or more epitopes within or thatinclude these residues and using such epitopes to generate antibodies(see Section VI below). Alternatively, peptides that spanned the regionsthat included amino acids 658-1114 or 1006-1114 of GenBank Accession No.NM_(—)020975, or peptides that included deletions or other mutations inthese regions, could be generated and tested as described above toidentify amino acids of the protein-protein interface (see Section IVabove). Crystallography with a peptide that included amino acids658-1114 or 1006-1114 of GenBank Accession No. NM_(—)020975 and a Retregulatory protein (see Table 3) could be performed to identify aminoacids of the protein-protein interface (see Section IV above).

A. EGFR Regulatory Domain Peptides

This disclosure concerns, among other things, the discovery of a19-amino acid region of EGFR that can be used, e.g., to interrogate thestructural and/or functional state of the receptor. For example, thisregion can be used to generate diagnostic specific binding reagents.This region has the sequence: LDNPDYQQDFFPKEAKPNG (SEQ ID NO: 2; “L2GPeptide”). It is found in the C-terminal, intracellular (or cytoplasmic)domain of EGFR (for exemplary EGFR sequences, see, e.g., GENBANK™Accession Nos. XP_(—)001156546.1; XP_(—)001156495.1; XP_(—)519102.2;XP_(—)001156439.1; BAD92679.1; AAS07524.1; AAX41033.1; NP_(—)113695.1;AAT52212.1; NP_(—)005219.2; and CAA25240.1).

The intracellular domain of EGFR, which corresponds to residues 669-1210of SEQ ID NO: 1, includes a kinase domain (residues 712-979 of SEQ IDNO: 1) and a regulatory domain (residues 980-1210). The EGFR regulatorydomain includes at least five tyrosine residues (Tyr1016, Tyr1092,Tyr1110, Tyr1172, and Tyr1197 of SEQ ID NO: 1), which are believed to beautophosphorylation sites (Chattopadhyay et al., J. Biol. Chem.,274:26091-26097, 1999). Among all of the C-terminal tyrosine residues,there are three YXXL/V and four YXXP/D motifs, which, for manytransmembrane receptors, serve as the docking sites for Src homology 2(SH2) domain-containing proteins (Xia et al., J. Biol. Chem.,277(34):30716-30723, 2002). As a class, SH2 domain-containing proteinsare accepted phosphorylation-dependent regulators of intracellularsignal cascades.

The EGFR regulatory domain contains an inhibitory subdomain (Xia et al.,J. Biol. Chem., 277(34):30716-30723, 2002), which corresponds toresidues 1138-1196 of SEQ ID NO: 1. The L2G Peptide sequence iscontained within this inhibitory subdomain. The inhibitory subdomain isbelieved at least to mediate a protein-protein interaction between EGFRand SOCS proteins (e.g., SOCS1 and SOCS3) (Xia et al., J. Biol. Chem.,277(34):30716-30723, 2002). The interaction between EGFR and SOCSproteins is further believed to stimulate the proteasomal degradation ofthe EGFR complex and/or induce degradation of EGFR-associated STATproteins and/or block EGFR from further recruitment and activation ofSTAT proteins (Xia et al., J. Biol. Chem., 277(34):30716-30723, 2002).In each instance, the SOCS protein (e.g., SOCS1 and/or SOCS3)interaction directly or indirectly inhibits EGFR activity.

As demonstrated in this disclosure, epitopes present in the L2G Peptidesequence of EGFR are inaccessible to cognate RD-binding molecules (e.g.,antibodies) in some normal or neoplastic tissues. The accessibility ofsuch epitope is restored in tissues that lack proteins that normallybind the EGFR regulatory domain and the EGFR inhibitory subdomain.Hence, the disclosed L2G Peptide and other RDPs derived therefrom areuseful, at least, to make RD-binding molecules (such as antibodies,antibody fragments, scaffold polypeptides including antibody bindingdomains and aptamers) that expose the structural and correspondingfunctional states of EGFR.

In one embodiment, a disclosed RDP is the L2G Peptide, which has thesequence LDNPDYQQDFFPKEAKPNG (SEQ ID NO: 2). Also contemplated in someembodiments are immunogenic fragments of the L2G Peptide, whichfragments can be useful for producing a disclosed RD-binding molecule.For example, as demonstrated in Example 4, at least the subsequenceQQDFFPK (residues 7-13 of SEQ ID NO: 2) is sufficient to produce adisclosed RD-binding molecule (e.g., monoclonal antibody). Thus, in someembodiments, an immunogenic fragment of SEQ ID NO: 2 is at least 7contiguous residues of SEQ ID NO: 2 and includes the sequence QQDFFPK(residues 7-13 of SEQ ID NO: 2). In more specific embodiments animmunogenic fragment of SEQ ID NO: 2 is between 7 and 18 contiguousresidues of SEQ ID NO: 2 and includes the sequence QQDFFPK (residues7-13 of SEQ ID NO: 2). In other specific embodiments an immunogenicfragment of SEQ ID NO: 2 is between 10 and 18 contiguous residues of SEQID NO: 2 and includes the sequence QQDFFPK (residues 7-13 of SEQ ID NO:2). In each instance an immunogenic fragment of SEQ ID NO: 2 has afunction described herein (see, e.g., Abbreviations and Terms) orotherwise known in the art.

Further, at least because the subsequence QQDFFPK (residues 7-13 of SEQID NO: 2) is sufficient to produce a disclosed antigen-binding molecule,other RDP embodiments have the consensus sequence X1-6QQDFFPKX7-12,where X1 through X12 are any amino acid. In more specific embodiments,an L2G peptide has the sequence X1-6QQDFFPKX7-12, where X1 through X12are any conservative substitution (e.g., very highly conservedsubstitution, highly conserved substitution or conserved substitution)of the corresponding amino acid residue in SEQ ID NO: 2. Exemplaryconservative amino acid substitutions are set forth in Table 6.

TABLE 6 Exemplary conservative amino acid substitutions. Very Highly -Highly Conserved Original Conserved Substitutions (from the ConservedSubstitutions Residue Substitutions Blosum90 Matrix) (from the Blosum65Matrix) Ala Ser Gly, Ser, Thr Cys, Gly, Ser, Thr, Val Arg Lys Gln, His,Lys Asn, Gln, Glu, His, Lys Asn Gln; His Asp, Gln, His, Lys, Ser, ThrArg, Asp, Gln, Glu, His, Lys, Ser, Thr Asp Glu Asn, Glu Asn, Gln, Glu,Ser Cys Ser None Ala Gln Asn Arg, Asn, Glu, His, Lys, Met Arg, Asn, Asp,Glu, His, Lys, Met, Ser Glu Asp Asp, Gln, Lys Arg, Asn, Asp, Gln, His,Lys, Ser Gly Pro Ala Ala, Ser His Asn; Gln Arg, Asn, Gln, Tyr Arg, Asn,Gln, Glu, Tyr Ile Leu; Val Leu, Met, Val Leu, Met, Phe, Val Leu Ile; ValIle, Met, Phe, Val Ile, Met, Phe, Val Lys Arg; Gln; Glu Arg, Asn, Gln,Glu Arg, Asn, Gln, Glu, Ser, Met Leu; Ile Gln, Ile, Leu, Val Gln, Ile,Leu, Phe, Val Phe Met; Leu; Tyr Leu, Trp, Tyr Ile, Leu, Met, Trp, TyrSer Thr Ala, Asn, Thr Ala, Asn, Asp, Gln, Glu, Gly, Lys, Thr Thr SerAla, Asn, Ser Ala, Asn, Ser, Val Trp Tyr Phe, Tyr Phe, Tyr Tyr Trp; PheHis, Phe, Trp His, Phe, Trp Val Ile; Leu Ile, Leu, Met Ala, Ile, Leu,Met, Thr

Some exemplary RDPs having the consensus sequence X1-6QQDFFPKX7-12,wherein any 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or 11 or all 12 of residuesX1 through X12 will have conservative amino acid changes (such as, veryhighly conserved substitutions, highly conserved substitutions orconserved substitutions) as compared to SEQ ID NO: 2 and, as applicable,the remaining residues will have no change as compared to SEQ ID NO: 2.

In other embodiments, a RDP is a sequence variant of an L2G Peptide thathas at least 99%, at least 98%, at least 95%, at least 92%, at least90%, at least 85%, at least 80%, at least 75%, at least 70%, at least65%, or at least 60% amino acid sequence identity to the amino acidsequence set forth in SEQ ID NO: 2. “Sequence identity” is a phrasecommonly used to describe the similarity between two amino acidsequences (or between two nucleic acid sequences). Sequence identitytypically is expressed in terms of percentage identity; the higher thepercentage, the more similar the two sequences.

Methods for aligning sequences for comparison and determining sequenceidentity are well known in the art. Various programs and alignmentalgorithms are described in: Smith and Waterman, Adv. Appl. Math.,2:482, 1981; Needleman and Wunsch, J. Mol. Biol., 48:443, 1970; Pearsonand Lipman, Proc. Natl. Acad. Sci. USA, 85:2444, 1988; Higgins andSharp, Gene, 73:237-244, 1988; Higgins and Sharp, CABIOS, 5:151-153,1989; Corpet et al., Nucleic Acids Research, 16:10881-10890, 1988;Huang, et al., Computer Applications in the Biosciences, 8:155-165,1992; Pearson et al., Methods in Molecular Biology, 24:307-331, 1994;Tatiana et al., FEMS Microbiol. Lett., 174:247-250, 1999. Altschul etal. present a detailed consideration of sequence-alignment methods andhomology calculations (J. Mol. Biol., 215:403-410, 1990).

The National Center for Biotechnology Information (NCBI) Basic LocalAlignment Search Tool (BLAST™, Altschul et al., J. Mol. Biol.,215:403-410, 1990) is publicly available from several sources, includingthe National Center for Biotechnology Information (NCBI, Bethesda, Md.)and on the Internet, for use in connection with the sequence-analysisprograms blastp, blastn, blastx, tblastn and tblastx. A description ofhow to determine sequence identity using this program is available onthe internet under the help section for BLAST™.

For comparisons of amino acid sequences of greater than about 15 aminoacids, the “Blast 2 sequences” function of the BLAST™ (Blastp) programis employed using the default BLOSUM62 matrix set to default parameters(cost to open a gap [default=5]; cost to extend a gap [default=2];penalty for a mismatch [default=3]; reward for a match [default=1];expectation value (E) [default=10.0]; word size [default=3]; and numberof one-line descriptions (V) [default=100]. When aligning short peptides(fewer than around 15 amino acids), the alignment should be performedusing the Blast 2 sequences function “Search for short nearly exactmatches” employing the PAM30 matrix set to default parameters (expectthreshold=20000, word size=2, gap costs: existence=9 and extension=1)using composition-based statistics.

Any disclosed sequence variant of a L2G Peptide (whether it is a varianthaving one or more conservative amino acid substitutions as compare toSEQ ID NO: 2 or a variant having a disclosed percentage sequenceidentity to SEQ ID NO: 2), at least, is immunogenic (alone or whencouple to a carrier molecule) and, e.g., capable of eliciting productionof a RD-binding molecule (such as a monoclonal antibody). Similarly, theprotein sequences associated with the GenBank Nos. listed in Table 5 forthe RTK ID, RD, and inhibitory domains can be engineered to include oneor more conservative amino acid substitutions (such as 1 to 5 or 1 to 10conservative amino acid substitutions), wherein the resulting variantretains RTK biological activity, and in some examples is immunogenic. Inother embodiments, the protein sequences associated with the RTK ID, RD,and inhibitory domains for the GenBank Nos. listed in Table 5 have atleast 99%, at least 98%, at least 95%, at least 92%, at least 90%, atleast 85%, at least 80%, at least 75%, at least 70%, at least 65%, or atleast 60% amino acid sequence identity to the amino acid sequence setforth in the GenBank Accession No. (which are herein all incorporated byreference), wherein the resulting variant retains RTK biologicalactivity, and in some examples is immunogenic.

VI. Generation of Diagnostic Specific Binding Reagents

This disclosure also concerns the generation of diagnostic specificbinding reagents, which are useful for determining the activity state ofan oncogenic RTK in a cell. For example, with knowledge of a region oramino acid residues of an interface between directly interactingproteins (e.g., between an RTK ID and a regulatory protein) or knowledgeof the location of an RTK ID or portion thereof (such as an RD or aninhibitory domain) a binding molecule that specifically recognizes theinterface region (e.g., a region of the RTK RD) or an epitope thatincludes at least a portion of the interface residues (i.e., aninterface-specific binding molecule) can be obtained from a commerciallyavailable source or prepared using techniques common in the art. Forexample, methods of preparing antibodies, antibody fragments, aptamersand other antigen-binding molecules are described in detail elsewhere inthis disclosure.

Such diagnostic specific binding molecules are a species ofantigen-binding molecules that can specifically bind to amino acids thatform a protein-protein interface between an RTK and a regulatory proteinin the absence of the RTK protein-regulatory protein interaction andfail to specifically bind amino acids that form a protein-proteininterface between an RTK and a regulatory protein in the presence of theRTK protein-regulatory protein interaction. In a specific example, suchspecific binding reagents specifically bind one or more epitopes in anRTK ID, such as an RD within the ID (see Table 5). It will beappreciated that a diagnostic specific binding molecule need not bindall amino acids of the protein-protein interface to achieve the desiredresult. For example, binding to even a single amino acid (or at leastone amino acid, such as 1 to 20, 1 to 10, or 2 to 10) of theprotein-protein interface (along with binding to other amino acids e.g.,of the RTK ID) may still permit the diagnostic specific binding moleculeto specifically bind to the protein-protein interface in the absence ofthe RTK protein-regulatory protein interaction and fail to specificallybind the protein-protein interface in the presence of the RTKprotein-regulatory protein interaction.

Exemplary diagnostic specific binding reagents include those specificfor an EGFR L2G peptide, or any epitope contained therein, or diagnosticspecific binding reagents specific for a L2G peptide sequence in EGFR(including specific binding molecules that are competitively inhibitedfrom binding EGFR by a L2G peptide or fragment thereof), diagnosticspecific binding reagents that recognize particular structural states ofEGFR (for instance, diagnostic specific binding reagents specific forepitopes masked by EGFR protein-protein interactions), and/or diagnosticspecific binding reagents that recognize particular regulated states ofEGFR (for instance, diagnostic specific binding reagents specific forepitopes contained with an EGFR inhibitory subdomain and which recognizethe binding of a negative regulatory molecule (e.g., SOCS1 or SOCS2) toEGFR.

Diagnostic specific binding reagents include, for example, antibodies orfunctional fragments or recombinant derivatives thereof, aptamers,mirror-image aptamers, or engineered nonimmunoglobulin binding proteinsbased on any one or more of the following scaffolds: fibronectin (e.g.,ADNECTINS™ or monobodies), CTLA-4 (e.g., EVIBODIES™), tendamistat (e.g.,McConnell and Hoess, J. Mol. Biol., 250:460-470, 1995), neocarzinostatin(e.g., Heyd et al., Biochem., 42:5674-5683, 2003), CBM4-2 (e.g.,Cicortas-Gunnarsson et al., Protein Eng. Des. Sel., 17:213-221, 2004),lipocalins (e.g., ANTICALINS™; Schlehuber and Skerra, Drug Discov.Today, 10:23-33, 2005), T-cell receptors (e.g., Chlewicki et al., J.Mol. Biol., 346:223-239, 2005), protein A domain (e.g., AFFIBODIES™;Engfeldt et al., ChemBioChem, 6:1043-1050, 2005), Im9 (e.g., Bernath etal., J. Mol. Biol., 345:1015-1026, 2005), ankyrin repeat proteins (e.g.,DARPins; Amstutz et al., J. Biol. Chem., 280:24715-24722, 2005),tetratricopeptide repeat proteins (e.g., Cortajarena et al., ProteinEng. Des. Sel., 17:399-409, 2004), zinc finger domains (e.g., Bianchi etal., J. Mol. Biol., 247:154-60, 1995), pVIII (e.g., Petrenko et al.,Protein Eng., 15:943-950, 2002), GCN4 (Sia and Kim, Proc. Natl. Acad.Sci. USA, 100:9756-61, 2003), avian pancreatic polypeptide (APP) (e.g.,Chin et al., Bioorg. Med. Chem. Lett., 11:1501-5, 2001), WW domains,(e.g., Dalby et al., Protein Sci., 9:2366-76, 2000), SH3 domains (e.g.,Hiipakka et al., J. Mol. Biol., 293:1097-106, 1999), SH2 domains(Malabarba et al., Oncogene, 20:5186-5194, 2001), PDZ domains (e.g.,TELOBODIES™; Schneider et al., Nat. Biotechnol., 17:170-5, 1999), TEM-1β-lactamase (e.g., Legendre et al., Protein Sci., 11:1506-18, 2002),green fluorescent protein (GFP) (e.g., Zeytun et al., Nat. Biotechnol.,22:601, 2004), thioredoxin (e.g., peptide aptamers; Lu et al.,Biotechnol., 13:366-372, 1995), Staphylococcal nuclease (e.g., Norman,et al., Science, 285:591-5, 1999), PHD fingers (e.g., Kwan et al.,Structure, 11:803-13, 2003), chymotrypsin inhibitor 2 (CI2) (e.g.,Karlsson et al., Br. J. Cancer, 91:1488-94, 2004), bovine pancreatictrypsin inhibitor (BPTI) (e.g., Roberts, Proc. Natl. Acad. Sci. USA,89:2429-33, 1992) and many others (see review by Binz et al., Nat.Biotechnol., 23(10):1257-68, 2005 and supplemental materials).

Disclosed diagnostic specific binding reagents also include aptamers. Inone example, an aptamer is a single-stranded nucleic acid molecule (suchas, DNA or RNA) that assumes a specific, sequence-dependent shape andbinds to a target protein (e.g., an RTK protein-regulatory proteininterface, for example a region of an RTK ID or RD that binds aregulatory protein) with high affinity and specificity. Aptamersgenerally comprise fewer than 100 nucleotides, fewer than 75nucleotides, or fewer than 50 nucleotides (such as 10 to 95 nucleotides,25 to 80 nucleotides, 30 to 75 nucleotides, or 25 to 50 nucleotides). Ina specific embodiment, a disclosed diagnostic specific binding reagentis a mirror-image aptamer (also called a SPIEGELMER™). Mirror-imageaptamers are high-affinity L-enantiomeric nucleic acids (for example,L-ribose or L-2′-deoxyribose units) that display high resistance toenzymatic degradation compared with D-oligonucleotides (such as,aptamers). The target binding properties of aptamers and mirror-imageaptamers are designed by an in vitro-selection process starting from arandom pool of oligonucleotides, as described for example, in Wlotzka etal., Proc. Natl. Acad. Sci. 99(13):8898-8902, 2002. Methods ofgenerating aptamers are known in the art (see e.g., Fitzwater andPolisky (Methods Enzymol., 267:275-301, 1996; Murphy et al., Nucl. AcidsRes. 31:e110, 2003).

In another example, an aptamer is a peptide aptamer that binds to atarget protein (e.g., an RTK protein-regulatory protein interface, forexample a region of an RTK ID or RD that binds a regulatory protein)with high affinity and specificity. Peptide aptamers include a peptideloop (e.g., which is specific for the RTK protein-regulatory proteininterface) attached at both ends to a protein scaffold. This doublestructural constraint greatly increases the binding affinity of thepeptide aptamer to levels comparable to an antibody's (nanomolar range).The variable loop length is typically 8 to 20 amino acids (e.g., 8 to 12amino acids), and the scaffold may be any protein which is stable,soluble, small, and non-toxic (e.g., thioredoxin-A, stefin A triplemutant, green fluorescent protein, eglin C, and cellular transcriptionfactor Sp1). Peptide aptamer selection can be made using differentsystems, such as the yeast two-hybrid system (e.g., Gal4yeast-two-hybrid system) or the LexA interaction trap system.

Disclosed diagnostic specific binding reagents also include antibodies.The term “antibody” refers to an immunoglobulin molecule (orcombinations thereof) that specifically binds to, or is immunologicallyreactive with, a particular antigen, and includes polyclonal,monoclonal, genetically engineered and otherwise modified forms ofantibodies, including but not limited to chimeric antibodies, humanizedantibodies, heteroconjugate antibodies (e.g., bispecific antibodies,diabodies, triabodies, and tetrabodies), single chain Fv antibodies(scFv), polypeptides that contain at least a portion of animmunoglobulin that is sufficient to confer specific antigen binding tothe polypeptide, and antigen binding fragments of antibodies. Antibodyfragments include proteolytic antibody fragments [such as F(ab′)2fragments, Fab′ fragments, Fab′-SH fragments, Fab fragments, Fv, andrIgG], recombinant antibody fragments (such as sFv fragments, dsFvfragments, bispecific sFv fragments, bispecific dsFv fragments,diabodies, and triabodies), complementarity determining region (CDR)fragments, camelid antibodies (see, for example, U.S. Pat. Nos.6,015,695; 6,005,079; 5,874,541; 5,840,526; 5,800,988; and 5,759,808),and antibodies produced by cartilaginous and bony fishes and isolatedbinding domains thereof (see, for example, International PatentApplication No. WO03014161).

A Fab fragment is a monovalent fragment consisting of the VL, VH, CL andCH1 domains; a F(ab′)₂ fragment is a bivalent fragment comprising twoFab fragments linked by a disulfide bridge at the hinge region; an Fdfragment consists of the VH and CH1 domains; an Fv fragment consists ofthe VL and VH domains of a single arm of an antibody; and a dAb fragmentconsists of a VH domain (see, e.g., Ward et al., Nature 341:544-546,1989). A single-chain antibody (scFv) is an antibody in which a VL andVH region are paired to form a monovalent molecule via a syntheticlinker that enables them to be made as a single protein chain (see,e.g., Bird et al., Science, 242: 423-426, 1988; Huston et al., Proc.Natl. Acad. Sci. USA, 85:5879-5883, 1988). Diabodies are bivalent,bispecific antibodies in which VH and VL domains are expressed on asingle polypeptide chain, but using a linker that is too short to allowfor pairing between the two domains on the same chain, thereby forcingthe domains to pair with complementary domains of another chain andcreating two antigen binding sites (see, e.g., Holliger et al., Proc.Natl. Acad. Sci. USA, 90:6444-6448, 1993; Poljak et al., Structure,2:1121-1123, 1994). A chimeric antibody is an antibody that contains oneor more regions from one antibody and one or more regions from one ormore other antibodies. An antibody may have one or more binding sites.If there is more than one binding site, the binding sites may beidentical to one another or may be different. For instance, a naturallyoccurring immunoglobulin has two identical binding sites, a single-chainantibody or Fab fragment has one binding site, while a “bispecific” or“bifunctional” antibody has two different binding sites.

As discussed above, exemplary diagnostic specific binding reagentsrecognize particular regulated or structural states of a target RTK. Forexample, a disclosed diagnostic specific binding reagent can detect themasking (or unmasking) of an epitope in an RTK ID or RD (such as theEGFR regulatory domain; residues 980-1210 of SEQ ID NO: 1, or other IDor RD listed in Table 5). Such epitope masking (or unmasking) canresult, for instance, from a protein-protein interaction between the RTKand another cellular protein (e.g., between EGFR and a SOCS protein suchas SOCS1 or SOCS3); wherein the binding of the cellular protein to theRTK masks the epitope and the disassociation (or lack of association) ofthe two proteins unmasks the epitope. Other examples involve diagnosticspecific binding reagents (such as antibodies (e.g., monoclonalantibody) or fragments thereof) that specifically bind to an RTK ID, RDor inhibitory domain; wherein such specific binding is competitivelyinhibited by a positive or negative regulatory protein, such as thoselisted in Table 3 (or a fragment of such regulatory proteins that bindsto the RTK ID, for example, a region of the RTK ID including aphosphorylated Tyr residue).

In some examples, diagnostic specific binding reagents, such asantibodies (e.g., monoclonal antibody) or fragments thereof, arecharacterized by specific binding to an RTK ID or RD (such as thosedisclosed herein, see, e.g., Table 5 and Section V). In other examples,diagnostic specific binding reagents, such as antibodies (e.g.,monoclonal antibody) or fragments thereof, specifically bind to aminoacid residues of EGFR that correspond to the sequence(s) of disclosedRDPs (see, e.g., Section V above). In still other examples, diagnosticspecific binding reagents, such as antibodies (e.g., monoclonalantibody) or fragments thereof, specifically bind to an RTK regulatorydomain (e.g., EGRF RD) or to an inhibitory subdomain (e.g., the EGFRinhibitory subdomain) and such specific binding is competitivelyinhibited by any one or more RDP (e.g., EGFR RDPs) disclosed herein(see, e.g., Section V). Other specific examples involve diagnosticspecific binding reagents (such as antibodies (e.g., monoclonalantibody) or fragments thereof) that specifically bind to an RTK (e.g.,the EGFR) RD or to an RTK (e.g., EGFR) inhibitory subdomain; whereinsuch specific binding is competitively inhibited by a regulatory protein(e.g., for EGFR the SOCS protein, such as SOCS1 or SOCS3, or a fragmentof a SOCS protein that binds to the regulatory domain of EGFR, forexample, a region of the regulatory domain including a phosphorylatedTyr residue).

In one particular embodiment, a diagnostic specific binding reagent is arabbit monoclonal antibody deposited at ATCC Accession No. ______. Inanother particular embodiment, a diagnostic specific binding reagent israbbit monoclonal antibody clone 5B7, which is commercially availablefrom Ventana Medical Systems (Tucson, Ariz.; product number 790 4347).

In some examples, an antibody specifically binds to a target (such as aprotein-protein interface, for example an RTK ID) with a bindingconstant that is at least 10³ M⁻¹ greater, 10⁴ M⁻¹ greater or 10⁵ M⁻¹greater than a binding constant for other molecules in a sample. In someexamples, a diagnostic specific binding reagent (such as an antibody(e.g., monoclonal antibody) or fragments thereof) has an equilibriumconstant (K_(d)) of 1 nM or less. For example, diagnostic specificbinding reagents are provided that bind to an RTK ID (such as aregulatory domain or inhibitory subdomain) with a binding affinity of atleast about 0.1×10⁻⁸ M, at least about 0.3×10⁻⁸ M, at least about0.5×10⁻⁸ M, at least about 0.75×10⁻⁸ M, at least about 1.0×10⁻⁸ M, atleast about 1.3×10⁻⁸ M at least about 1.5×10⁻⁸M, or at least about2.0×10⁻⁸ M. Kd values can, for example, be determined by competitiveELISA (enzyme-linked immunosorbent assay) or using a surface-plasmonresonance device such as the Biacore T100, which is available fromBiacore, Inc., Piscataway, N.J.

A disclosed diagnostic specific binding reagent, such as an antibody(e.g., monoclonal antibody), aptamer, or fragments thereof, optionallycan be directly labeled with a detectable moiety. Useful detectionagents include fluorescent compounds (including fluorescein, fluoresceinisothiocyanate, rhodamine, 5-dimethylamine-1-napthalenesulfonylchloride, phycoerythrin, lanthanide phosphors, or the cyanine family ofdyes (such as Cy-3 or Cy-5) and the like); bioluminescent compounds(such as luciferase, green fluorescent protein (GFP), or yellowfluorescent protein); enzymes that can produce a detectable reactionproduct (such as horseradish peroxidase, β-galactosidase, luciferase,alkaline phosphatase, or glucose oxidase and the like), or radiolabels(such as ³H, ¹⁴C, ¹⁵N, ³⁵S, ⁹⁰Y, ⁹⁹Tc, ¹¹¹In, ¹²⁵I, or ¹³¹I).

A. Making of Exemplary Diagnostic Antibodies

Methods of making diagnostic specific binding molecules are well knownin the art. The method used will depend upon the nature of the desiredbinding molecules; for instance peptide-based diagnostic specificbinding molecules that are not necessarily immunoglobulin in origin canbe made using methods that are similar to phage display methods. Onesuch method is described in Szardenings, J. Recept. Signal Transduct.Res., 23:307-309, 2003.

Methods of generating antibodies (such as monoclonal or polyclonalantibodies) are well established in the art (for example see Harlow andLane, Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory,New York, 1988). For example identified peptide fragments of an RTKprotein-regulatory protein interface (such as a region(s) of an RTK IDor RD or inhibitory domain) (see Table 5 and Section V) can beconjugated to carrier molecules (or nucleic acids encoding such epitopesor conjugated RDPs) can be injected into non-human mammals (such as miceor rabbits), followed by boost injections, to produce an antibodyresponse. Serum isolated from immunized animals may be isolated for thepolyclonal antibodies contained therein, or spleens from immunizedanimals may be used for the production of hybridomas and monoclonalantibodies. In some examples, antibodies are purified before use.

In one example, monoclonal antibody to an epitope of an RTKprotein-regulatory protein interface (e.g., an epitope of an RTK ID, RD,or inhibitory domain) can be prepared from murine hybridomas accordingto the classical method of Kohler and Milstein (Nature, 256:495, 1975)or derivative methods thereof. Briefly, a mouse (such as Balb/c) isrepetitively inoculated with a few micrograms of the selected peptidefragment (e.g., epitope of an RTK ID, such as SEQ ID NO: 2) or carrierconjugate thereof over a period of a few weeks. The mouse is thensacrificed, and the antibody-producing cells of the spleen isolated. Thespleen cells are fused by means of polyethylene glycol with mousemyeloma cells, and the excess unfused cells destroyed by growth of thesystem on selective media comprising aminopterin (HAT media). Thesuccessfully fused cells are diluted and aliquots of the dilution placedin wells of a microtiter plate where growth of the culture is continued.Antibody-producing clones are identified by detection of antibody in thesupernatant fluid of the wells by immunoassay procedures, such as ELISA,as originally described by Engvall (Enzymol., 70:419, 1980), andderivative methods thereof. Selected positive clones can be expanded andtheir monoclonal antibody product harvested for use.

In another example, monoclonal antibody to epitopes of an RTKprotein-regulatory protein interface (e.g., an epitope of an RTK ID, RD,or inhibitory domain) can be prepared from rabbit hybridomas asdescribed in U.S. Pat. Nos. 7,148,332, 5,675,063, or 4,859,595.

In yet another example, monoclonal antibodies to epitopes of an RTKprotein-regulatory protein interface (e.g., an epitope of an RTK ID, RD,or inhibitory domain) can be prepared by repetitively inoculating anon-human mammal (such as a mouse or rabbit) with one or more plasmidsencoding a disclosed RDP (such as a plasmid encoding SEQ ID NO: 2). Forexample, pcDNA3 (Invitrogen, Carlsbad, Calif.) or a vector derived therefrom, can be manipulated using standard molecular biology methods toinclude a coding sequence for a disclosed peptide fragment of an RTKprotein-regulatory protein interface (e.g., SEQ ID NO: 2). In oneexemplary method, Balb/c mice (6-8 weeks old) are immunized three timeswith the appropriate plasmid (20 μg in phosphate-buffered saline), andone boost can be given with cells before fusion. Mice can be injectedthree times intradermally into the base of the tail on days 0, 10, and20 using an insulin syringe with a 28-gauge needle attached. Serum canbe drawn on days 30 and 45 for evaluation of the anti-serum titer. Toboost the immunized mice, cells expressing the desired plasmid areinjected (for example on day at least 50). These injections can beintravenous and intraperitoneal. Spleens are harvested about 80-90 hoursafter the last cell boost for cell fusion.

Cell fusions of the splenocytes can be performed according to theprotocol of Oi and Herzenberg (Selected Methods in Cellular Immunology,Freeman Press, San Francisco, 1980). Splenocytes and SP2/0 cells aremixed, for example at a 4:1 ratio. The mixed cells are centrifuged andthe cell pellet resuspended in polyethylene glycol (such as 40%-50%(w/v) polyethylene glycol) and appropriate medium. The resultingsuspension is centrifuged and the cell pellet resuspended in HAT medium,and seeded in 96-well plates at 100 μl/well (2.5×10⁵ cells/well) andcultured in a CO₂ incubator. On the day after fusion, 100 μl of freshHAT medium containing 500 μg/ml geneticin (Invitrogen) is added. On days4 and 7, half of the spent medium is replaced by fresh HAT mediumcontaining 250 μg/ml geneticin. On day 8, the growth of the hybridoma ineach well is checked under a microscope. mAb production in culturesupernatants can be assayed on day 10 by ELISA assay or days 9 and 10 byFACS sorter. Positive clones can be expanded and the specific hybridomascloned by a limiting dilution method.

In addition, protocols for producing humanized forms of monoclonalantibodies and fragments of monoclonal antibodies are known in the art(see, e.g., U.S. Pat. Nos. 6,054,297, 6,407,213, 6,639,055, 6,800,738,and 6,719,971 and U.S. Pat. Appl. Pub. Nos. 2005/0033031, and2004/0236078). Similarly, methods for producing single chain antibodieshave been described and can be useful for the making of diagnosticspecific binding molecules disclosed herein (see, Buchner et al., Anal.Biochem. 205:263-270, 1992; Pluckthun, Biotechnology 9:545, 1991; Huseet al., Science 246:1275, 1989 and Ward et al., Nature 341:544, 1989).

B. Making of Exemplary Diagnostic Aptamers

Methods of generating aptamers (e.g., DNA or RNA aptamers) are wellestablished in the art. For example, with knowledge of an RTK IDsequence (such as a RD or inhibitory domain sequence, see Section V), orupon identification of at least 3 or at least 5 amino acid residues thatform a protein-protein interface when a regulatory protein is bound toan RTK ID (see Section IV), aptamers can be selected that bind to aminoacids of such an interface (such as an RTK ID or RD) in the absence of aregulatory protein binding to the RTK, but do not specifically bind toamino acids of such an interface (such as an RTK ID or RD) when aregulatory protein is bound to an RTK.

In one example, DNA or RNA aptamers are selected using the in vitromethod SELEX (systematic evolution of ligands by exponentialenrichment), for example using the method of Fitzwater and Polisky(Methods Enzymol., 267:275-301, 1996). Such a method can be used toidentify aptamers that bind with high specificity to an RTKprotein-regulatory protein interface, such as that bind specifically toa region of an RTK ID or RD that binds a regulatory protein. The SELEXprocedure is usually initiated with an RNA or DNA library containingabout 10¹⁴-10¹⁵ random oligonucleotide sequences. In a fully randomizedoligonucleotide library, each molecule will exhibit a unique tertiarystructure that will be dependent on the nucleotide sequence of thatmolecule. The binding affinity of the oligonucleotide for a particularprotein-protein interface will be determined by the fit between moietieson the surface of the oligonucleotide and epitopes on the targetprotein-protein interface. By starting from a library of vast diversity,aptamers of nanomolar or subnanomolar affinity for the targetprotein-protein interface with selectivity for that targetprotein-protein interface over other protein-protein interfaces with ahigh degree of structural homology can be identified. For example,peptides comprising the amino acids that form the target protein-proteininterface (or portion thereof, such as 3 to 20 amino acids of aprotein-protein interface, for example an epitope) can be attached to asurface (such as a 96-well or other multi-well microtiter plate). Thelibrary of nucleic acid molecules can be added to the bound peptideunder conditions that permit members of the library to bind to thepeptide (e.g., by incubating at 37° C. for 30 minutes). Unbound membersof the library are washed away, and then bound members of the libraryare eluted (e.g., by incubating at 95° C. for 10 minutes). Reversetranscription is performed (if the aptamers are RNA), followed bypolymerase chain reaction and transcription to generate nucleic acidsfor the next round of SELEX. The dissociation constant (Kd) forresulting selected aptamer can be determined using routine methods.Aptamers with high affinity for the desired protein-protein interfacecan be selected, such as a Kd of less than 100 nM, such as less than 50nM, less than 10 nM, or less than 1 nM (for example 0.1 to 50 nM).Aptamers can be modified to increase their half-life, for examplemodified with 2′-fluorine-substituted pyrimidines, 2′-ribo purines,polyethylene glycol (PEG) linkage, and the like.

In one example, peptide aptamers are selected using a yeast two hybridsystem, for example using the method of de Chassey et al. (Molecular &Cellular Proteomics 6:451-9, 2007). Reviews are provided, for example,in Borghouts et al. (Comb. Chem. High Throughput Screen. 11:135-45,2008) and Buerger et al. (J. Cancer Res. Clin. Oncol. 129:669-75, 2003).Such a method can be used to identify peptide aptamers that bind withhigh specificity to an RTK protein-regulatory protein interface, such asthat bind specifically to a region of an RTK ID or RD that binds aregulatory protein. A peptide aptamer library of high complexity isscreened, such as 20mer or 8-12mer libraries. The library may or may notbe based on information known about the sequence of the RTKprotein-regulatory protein interface. In one example, the libraryincludes oligonucleotides encoding variant peptides based on the aminoacid sequence of the RTK protein-regulatory protein interface. Thelibrary includes oligonucleotides encoding the variable peptidesinserted into a vector encoding the scaffold protein (e.g.,thioredoxin). When expressed, “prey” peptide sequences are embedded inthe scaffold protein. A nucleic acid sequence or vector encoding the“bait” target protein (e.g., sequence of the RTK protein-regulatoryprotein interface) fused to a transcription module (e.g., Gal4 or LexA)is expressed in the cells (e.g., yeast) along with the “prey” codingsequences.

If the yeast-two-hybrid system is used, the “prey” peptide aptamer canbe fused to Gal4-transactivation domain (Gal4-AD) and can also include anuclear localization signal and an HA tag for detection. Exemplaryvectors that can be used to express the peptide aptamer include pRS424,pAD-Trx, pGAD424; pGAD-T7, pACT2, and pAD-Gal4-2.1. A vector encodingthe “bait” target protein fused to the Gal4 DNA binding domain isexpressed in yeast along with the “prey” coding sequences. Exemplaryvectors that can be used to express the “bait” peptide include pPC97,pLex9, pGBK-T7, and pDB-Gal4Cam. In some examples, the reporter yeaststrain into which prey and bait vectors are introduced include His3,Ade2, Ura3 and LacZ genes under the control of a Gal upstream activatingsequences to permit selection of clones where the bait and preyspecifically bind. To select for desired peptide aptamers, transformedyeast cells are placed on media lacking histidine, adenine, or uracil.β-gal assays can be performed to quantify binding between identifiedaptamers and the target. To increase selection stringency, the amount of3-AT inhibitor (e.g., 10-100 mM) can be increased. Cells that growindicate the presence of peptide aptamer binding to the target RTKprotein-regulatory protein interface.

If the LexA interaction trap system is used, a vector encoding the“prey” peptide aptamer fused to B42 or B112 acid transactivation domaincan be used. Exemplary vectors that can be used to express the peptideaptamer include pWP1, pWP2, pJG4-5, pJM1, pHA3). A vector encoding the“bait” target protein fused to the DNA binding domain of the LexArepressor is expressed in yeast along with the “prey” coding sequences.An exemplary vector that can be used to express the “bait” peptideincludes pEG202. Expression of the prey vector is induced if galactoseis present in the growth medium. To select for desired peptide aptamers,transformed yeast cells are placed on media with galactose. Interactionsbetween bait protein and peptide aptamer are detected on galactoseplates that lack leucine. Cells that grow indicate the presence ofpeptide aptamer binding to the target RTK protein-regulatory proteininterface.

Clones indicated to carry the desired protein aptamer that binds to theRTK protein-regulatory protein interface can be selected, and the vectorencoding the aptamer isolated and cloned using standard recombinanttechnology.

C. Screening Diagnostic Agents

The diagnostic specific binding reagents generated (e.g., antibodies oraptamers) can be screened for their ability to specifically bind toamino acids that form a protein-protein interface between an RTK and aregulatory protein in the absence of the RTK protein-regulatory proteininteraction and fail to specifically bind amino acids that form aprotein-protein interface between an RTK and a regulatory protein in thepresence of the RTK protein-regulatory protein interaction. For example,a plurality of diagnostic specific binding reagents can be screened toidentify those that are excluded from binding when the protein-proteininterface is present, but bind with high specificity when theprotein-protein interface is absent. Such methods of screening areroutine in the art.

In one example, cells that express the target RTK but are functionallydeleted for the target regulatory protein associated with the diagnosticreagent are incubated with one or more of the diagnostic specificbinding reagents. For example, cells that do not express the regulatoryprotein due to a lack of transcription or translation of the geneencoding the regulatory protein (e.g., the regulatory protein gene isknocked out or the expression of the gene is inhibited using RNAitechnology, such as siRNA) can be used. Methods of making such cells areroutine in the art. In one example, the diagnostic specific bindingreagent is generated to determine if the negative regulator CBL wasbound to RON, and the cell used to screen the diagnostic reagentsexpresses RON but does not express functional CBL protein. Afterincubation of the functionally deleted cells with the one or morediagnostic specific binding reagents under conditions that would permitbinding of the regulatory protein to the RTK, the ability of thediagnostic specific binding reagents to bind to the RTK is determined,for example using western blotting, immunoprecipitation, pull-downassays, IHC and the like. The diagnostic specific binding reagents caninclude a label to permit detection if desired. Diagnostic specificbinding reagents that bind with high specificity to the RTK in cellsthat are functionally deleted for the target regulatory protein, but donot substantially bind the RTK in cells that express the targetregulatory protein, can be selected for further analysis.

In another example, an ELISA (enzyme-linked immunosorbent assay) is usedto screen the plurality of diagnostic specific binding reagents. Forexample, the target RTK-regulatory protein complex or the RTK proteinalone (or a portion thereof, such as the ID or RD) can be affixed to asolid surface (e.g., microtiter plate or nitrocellulose). In someexamples the RTK-regulatory complex is fixed to avoid competition withthe diagnostic specific binding reagents for the binding site on theRTK. After incubation of the protein-containing surfaces with the one ormore diagnostic specific binding reagents under conditions that wouldpermit binding of the regulatory protein to the RTK in the absence ofthe regulatory protein, the ability of the diagnostic specific bindingreagents to bind to the RTK is determined, for example by detecting alabel on the diagnostic specific binding reagents. Diagnostic specificbinding reagents that bind to the RTK in the absence of the regulatoryprotein, but do not bind the RTK-regulatory complexes, can be selectedfor further analysis.

In some examples, the plurality of diagnostic specific binding reagentsare screened to identify those having a low dissociation constant(K_(d)), and thus a high affinity, for the desired protein-proteininterface (e.g., the desired RTK ID or RD). Kd values can, for example,be determined by methods known in the art, such as competitive ELISA orusing a surface-plasmon resonance device such as the Biacore T100, whichis available from Biacore, Inc., Piscataway, N.J. In particularexamples, diagnostic specific binding reagents such as antibodies oraptamers are selected if they have a K_(d) of less than 100 nM, such asless than 50 nM, less than 10 nM, or less than 1 nM (for example 0.1 to50 nM). In some examples, a diagnostic specific binding reagent (such asan antibody (e.g., monoclonal antibody) or fragments thereof) has K_(d)of 1 nM or less.

D. Determining Diagnostic Value of Identified Specific Binding Reagents

In some examples, diagnostic specific binding reagents selected usingthe methods described above are analyzed for their diagnostic ability,such as the ability to determine if a subject has a cancer expressing atarget RTK, predict a disease outcome (such as disease-free survival),and to predict a subject's response to a therapy (such as an RTKinhibitory therapy). For example, diagnostic specific binding reagentsselected for their ability to specifically bind to amino acids that forma protein-protein interface between an RTK and a regulatory protein inthe absence of the RTK protein-regulatory protein interaction and failto specifically bind amino acids that form a protein-protein interfacebetween an RTK and a regulatory protein in the presence of the RTKprotein-regulatory protein interaction can be further analyzed for theirability to diagnose a subject (e.g., be used as a companion diagnostic,prognostic, or predictor).

In one example, a plurality of selected diagnostic specific bindingreagents are analyzed for their ability to be used as a diagnostic.Biological samples (e.g., tissue samples, such as fixed biopsy samples)from a cohort of subjects are subjected to IHC or other methods known inthe art (see Examples below) to permit determination of the activationstatus of the RTK using each of the selected diagnostic specific bindingreagents. For example, the subjects can be those known to have (or had)a tumor (e.g., cancer) expressing the target RTK.

To determine if the selected diagnostic specific binding reagent canfunction as a companion diagnostic, subject samples with a knownsurvival outcome can be analyzed. For example, such subjects can bethose who had a cancer expressing the target RTK, or had a cancer thatdid not express target RTK. A plurality of samples for each group (e.g.,+/− RTK expression) can be tested. In some examples, at least 50 or atleast 100 samples are analyzed for each test group. Each sample stainedwith a selected diagnostic specific binding reagent can be scored, forexample using the Tables provided in the Examples below (e.g., seeExample 12), and the results plotted (e.g., +/− RTK expression).Diagnostic specific binding reagents that have the ability to correlatepatients by their RTK expression status (e.g., +/− RTK expression), areindicated to be useful companion diagnostics and can be selected forclinical use.

To determine if the selected diagnostic specific binding reagent canfunction as a prognostic, subject samples with a known survival outcomecan be analyzed. For example, such subjects can be those who had acancer expressing the target RTK, did not receive an RTK-based therapy,and their time of survival after diagnosis known. A plurality of samplesfor each survival range (e.g., at least 6-months, at least 1-year, or atleast 5-years) can be tested. In some examples, at least 50 or at least100 samples are analyzed for each test group. Each sample stained with aselected diagnostic specific binding reagent can be scored, for exampleusing the Tables provided in the Examples below (e.g., see Example 12),and the results plotted (e.g., fraction survived versus time).Diagnostic specific binding reagents that have the ability to correlatepatient overall survivability (e.g., the ability to distinguish patientshaving poor survivability from those with greater survivability), areindicated to be useful in prognosing a subject and can be selected forclinical use.

To determine if the selected diagnostic specific binding reagent canfunction as a predictor, subject samples with a known response to an RTKtargeted therapy (such as an RTK inhibitory therapy, for example treatedwith a TKI) are analyzed. For example, such subjects can be those whohad a cancer expressing the target RTK, received an RTK-based therapy,and their time of survival after the therapy (or other response totherapy) known. A plurality of samples for each response (e.g., survivalof at least 6-months, at least 1-year, or at least 5-years, a decreasein tumor growth, volume or metastasis of particular relative amounts,such as decreases of at least 20%, at least 50%, or at least 90%) can betested. In some examples, at least 50 or at least 100 samples areanalyzed for each test group. Each sample stained with a selecteddiagnostic specific binding reagent can be scored, for example using theTables provided in the Examples below (e.g., see Example 12), and theresults plotted (e.g., fraction survived versus time or decrease intumor growth versus time). Diagnostic specific binding reagents thathave the ability to correlate patient overall survivability or otherresponse to the RTK therapy (e.g., the ability to distinguish patientsthat responded to the RTK-targeted therapy from those that that did notrespond to the RTK-targeted therapy), are indicated to be useful as apredictive agent can be selected for clinical use.

VII. Detecting Molecular Interactions in a Biological Sample

The identification and/or generation of diagnostic specific bindingreagents permits use of such agents to determine if particularprotein-protein interactions are present in a biological sample (e.g.,isolated cells or tissues). For example, the disclosed diagnosticspecific binding reagents can be used to determine the activation statusof an RTK, the status of which can be used to determine if a subject hasa particular RTK-expressing tumor, the prognosis of a subject having atumor, as well as the likelihood that the subject having a tumor willrespond to a particular RTK therapy. In some examples, the sample to beanalyzed using the disclosed diagnostic specific binding reagents ismounted on a solid surface (e.g., a microscope slide) and treated (e.g.,formalin-fixed and paraffin-embedded (“FFPE”)) to substantially maintainthe positions of components (e.g., proteins, RNAs and/or DNA) within thesample relative to one another.

Molecular interactions (e.g., protein-protein interactions) previouslyhave been studied in solution and using in vivo techniques, such asco-immunoprecipitation assays (where a protein of interest is capturedwith an antibody and any interaction partners bound to the protein aresubsequently identified by Western blot); pull-down assays (which aresimilar to co-immunoprecipitation assays, but use some ligand other thanan antibody to capture the protein complex); label transfer (where aknown protein is tagged with a detectable label and the label is thenpassed to an interacting protein); in vivo crosslinking of proteincomplexes (where cells are grown under conditions that causephotoreactive diazirine amino acid analogs to be incorporated intocellular proteins, which diazirines can be activated and bind tointeracting proteins); the yeast two-hybrid screen (which investigatesthe interaction between artificial fusion proteins inside the nucleus ofyeast); and dual polarisation interferometry (“DPI”) (which providesreal-time, high-resolution measurements of molecular size, density andmass). Each of the foregoing methods requires means to isolate (whetherphysically, chemically or otherwise) the components having a specificinteraction with one another from other non-interacting components.

Non-specific crosslinking reactions (such as, chemical crosslinking)also may be useful to examine protein-protein interactions in settingswhere non-specific interactions between reaction components can becontrolled. However, biological samples (e.g., isolated cells ortissues) mounted on a solid surface (e.g., microscope slides or supportmembranes) do not offer such a setting. Under those conditions,non-specific crosslinkers bond together (permanently orsemi-permanently) any components in the sample that in proximity of eachother whether or not such components interact under biologicalconditions.

Rather than view non-specific crosslinking as a hindrance to examiningprotein-protein interactions, the present disclosure actually exploitsthe non-specific crosslinking of biological components within a fixedbiological sample (e.g., FFPE cells or FFPE tissues). Such crosslinkingsubstantially ensures that the structural relationship betweeninteracting components in the sample (e.g., protein-protein orprotein-nucleic acid) is permanently or semi-permanently maintained;thereby, masking some or all residues that form the interface betweenthe components. For example, any epitope present in the interface wouldnot be available to a cognate antigen-binding protein (e.g., antibody)following fixation of the sample. Accordingly, the accessibility (ornot) of the residues within the interface to binding proteins (e.g.,antibodies) specific for such residues can be used to determine whetheror not the particular components were interacting in the biologicalsample at the time it was fixed. Therefore, by using fixed samples, therelationship between the RTK and a regulatory protein can be “frozen” intime and space (i.e., the presence of absence of a protein-proteininterface is fixed), and as a result, a disclosed diagnostic specificbinding reagent cannot compete for its binding site in theprotein-protein interface. In a non-fixed system, the binding of aregulatory protein to an RTK might not be detected as the diagnosticspecific binding reagent could “knock-off” the regulator from the RTKdue to competitive binding. Thus, the ability to determine theactivation status of the RTK would be lost.

Some disclosed methods involve identifying at least two biologicalcomponents (e.g., two proteins) that together form a direct interaction,determining the residues (e.g., amino acids) involved in the interfacebetween the at least two components, identifying or making at least onebinding molecule (such as a monoclonal antibody or fragment thereof)specific for some or all of the residues involved in the interfacebetween the at least two components, detecting in a fixed biologicalsample (such as FFPE tissue sections or fixed cell samples) the binding(or absence of binding) of the at least one interface-specificantigen-binding molecule. In some methods, the interacting componentsand the particular residues (or regions) involved in the interfacebetween the at least two components are known; hence, identifying suchcomponents and the nature of their interface would be optional steps ofthe disclosed method.

Because fixation of the interface(s) between the at least twointeracting components (e.g., proteins) leads to the exclusion ofdiagnostic specific binding reagents (e.g., interface-specificantigen-binding molecules) from binding residues in the interface(s),some methods will involve a negative result (i.e., no binding). In somesuch methods, it can be advantageous to further detect the presence ofone or more (e.g., one or two) components of the interaction complex;thus, showing that the failure of the at least one interface-specificantigen-binding molecule(s) to bind its target(s) is not due to absenceof one or more of the components involved in the making of theinterface(s) but rather is due to the masking of the target(s).

Biological samples useful in a disclosed method are isolated and includeany cell preparation or tissue preparation that can be fixed and mountedon a solid surface. Exemplary samples include, without limitation, bloodsmears, cytocentrifuge preparations, cytology smears, core biopsies,fine-needle aspirates, and/or tissue sections (e.g., cryostat tissuesections and/or paraffin-embedded tissue sections). Exemplary biologicalsamples may be isolated from normal cells or tissues, or from neoplasticcells or tissues. Neoplasia is a biological condition in which one ormore cells have undergone characteristic anaplasia with loss ofdifferentiation, increased rate of growth, invasion of surroundingtissue, and which cells may be capable of metastasis. Exemplaryneoplastic cells or tissues may be isolated from solid tumors, includingbreast carcinomas (e.g. lobular and duct carcinomas), sarcomas,carcinomas of the lung (e.g., non-small cell carcinoma, large cellcarcinoma, squamous carcinoma, and adenocarcinoma), mesothelioma of thelung, colorectal adenocarcinoma, stomach carcinoma, prostaticadenocarcinoma, ovarian carcinoma (such as serous cystadenocarcinoma andmucinous cystadenocarcinoma), ovarian germ cell tumors, testicularcarcinomas and germ cell tumors, pancreatic adenocarcinoma, biliaryadenocarcinoma, hepatocellular carcinoma, bladder carcinoma (including,for instance, transitional cell carcinoma, adenocarcinoma, and squamouscarcinoma), renal cell adenocarcinoma, endometrial carcinomas(including, e.g., adenocarcinomas and mixed Mullerian tumors(carcinosarcomas)), carcinomas of the endocervix, ectocervix, and vagina(such as adenocarcinoma and squamous carcinoma of each of same), tumorsof the skin (e.g., squamous cell carcinoma, basal cell carcinoma,melanoma, and skin appendage tumors), esophageal carcinoma, carcinomasof the nasopharynx and oropharynx (including squamous carcinoma andadenocarcinomas of same), salivary gland carcinomas, brain and centralnervous system tumors (including, for example, tumors of glial,neuronal, and meningeal origin), tumors of peripheral nerve, soft tissuesarcomas and sarcomas of bone and cartilage.

A solid support useful in a disclosed method need only bear thebiological sample and, optionally, but advantageously, permit theconvenient detection of components (e.g., proteins and/or nucleic acidsequences) in the sample. Exemplary supports include microscope slides(e.g., glass microscope slides or plastic microscope slides), coverslips(e.g., glass coverslips or plastic coverslips), tissue culture dishes,multi-well plates, membranes (e.g., nitrocellulose or polyvinylidenefluoride (PVDF)) or BIACORE™ chips.

Fixatives for mounted cell and tissue preparations are well known in theart and include, without limitation, 95% alcoholic Bouin's fixative; 95%alcohol fixative; B5 fixative, Bouin's fixative, formalin fixative,Karnovsky's fixative (glutaraldehyde), Hartman's fixative, Hollande'sfixative, Orth's solution (dichromate fixative), and Zenker's fixative(see, e.g., Carson, Histotechology: A Self-Instructional Text,Chicago:ASCP Press, 1997).

In some examples, the method includes detecting in a fixed biologicalsample a protein complex that includes (or consists of) a target RTK andan RTK-interacting protein (e.g., a regulatory protein). Specificexamples of known RTK-regulatory protein protein-protein interactionsare listed above in Table 3. In a specific example, the RTK is EGFR, andthe method includes detecting in a fixed biological sample a proteincomplex that includes (or consists of) EGFR and an EGFR-interactingprotein (e.g., a regulatory protein, such as a SOCS protein like SOCS1or SOCS3). EGFR is known to form protein-protein interactions in vivoand in vitro with numerous other proteins. Some such interactions arelisted in Table 7.

TABLE 7 Exemplary EGFR Interaction Partners PARTNER PUBMED 1 PARTNER 2SYSTEM SOURCE ID AREG EGFR In vivo Wong L et al. 10085134 CD44 EGFR Invivo Tsatas D et al. 12093135 EGFR GRB2 In vivo Lowenstein EJ et al.1322798 EGFR GRB2 In vivo Okutani T et al. 7527043 EGFR GRB2 In vitroLowenstein EJ et al. 1322798 EGFR GRB2 In vitro Okutani T et al. 7527043EGFR CTNNB1 In vivo Takahashi K et al. 9233779 EGFR CDC25A In vivo WangZ et al. 11912208 EGFR CDC25A In vitro Wang Z et al. 11912208 DCN EGFRIn vivo Santra M et al. 12105206 DCN EGFR In vivo Iozzo RV et al.9988678 DCN EGFR In vitro Santra M et al. 12105206 DCN EGFR In vitroIozzo RV et al. 9988678 DCN EGFR Two- Santra M et al. 12105206 hybridDCN EGFR Two- Iozzo RV et al. 9988678 hybrid EGFR HBEGF In vivo Shin SYet al. 12725245 EGF EGFR In vitro Stortelers C et al. 12093292 EGFR CAV1In vivo Couet J et al. 9374534 EGFR CAV1 In vitro Couet J et al. 9374534EGFR PRKACA In vivo Tortora G et al. 9050991 EGFR ERBB3 In vivo MarquesMM et al. 10527633 EGFR SHC1 In vitro Sakaguchi K et al. 9544989 ITGA5EGFR In vitro Kuwada SK et al. 10888683 ITGA5 EGFR In vivo Kuwada SK etal. 10888683 EGFR ZNF259 In vivo Moores SL et al. 10938113 EGFR RASA1 Invitro Serth J et al. 1633149 PTK2 EGFR In vivo Sieg DJ et al. 10806474PTK2 EGFR In vitro Sieg DJ et al. 10806474 PLSCR1 EGFR In vivo Sun J etal. 12009895 PLSCR1 EGFR In vivo Nanjundan M et al. 12871937 PLSCR1 EGFRIn vitro Sun J et al. 12009895 PLSCR1 EGFR In vitro Nanjundan M et al.12871937 GRB14 EGFR In vivo Daly RJ et al. 8647858 GRB14 EGFR In vitroDaly RJ et al. 8647858 EGFR DOK2 In vitro Jones N et al. 10508618 KRT7EGFR In vivo Blagoev B et al. 12577067 KRT7 EGFR In vitro Blagoev B etal. 12577067 TGFA EGFR In vitro Garrett TP et al. 12297049 EGFR XRCC6 Invivo Bandyopadhyay D 9430697 et al. EGFR PLD2 In vitro Slaaby R et al.9837959 EGFR PLD2 In vivo Slaaby R et al. 9837959 CD82 EGFR In vivoOdintsova E et al. 10985391 CD82 EGFR In vitro Odintsova E et al.10985391 SNX6 EGFR In vivo Parks WT et al. 11279102 SNX6 EGFR In vitroParks WT et al. 11279102 EGFR PTK2B In vivo Keely SJ et al. 10777553GRB10 EGFR In vivo Frantz JD et al. 9006901 GRB10 EGFR In vivo He W etal. 9506989 GRB10 EGFR In vitro Frantz JD et al. 9006901 GRB10 EGFR Invitro He W et al. 9506989 PRKCA EGFR In vivo Gauthier ML et al. 12878187PRKCA EGFR In vitro Gauthier ML et al. 12878187 EGFR STAT1 In vivo Xia Let al. 12070153 EGFR STAT1 In vitro Xia L et al. 12070153 KRT18 EGFR Invivo Blagoev B et al. 12577067 KRT18 EGFR In vitro Blagoev B et al.12577067 EGFR SOCS3 In vivo Xia L et al. 12070153 EGFR SOCS3 In vitroXia L et al. 12070153 EGFR MIG-6 In vivo Hackel PO et al. 11843178 EGFRMIG-6 Two- Hackel PO et al. 11843178 hybrid EGFR SOCS1 In vivo Xia L etal. 12070153 PLEC1 EGFR In vivo Blagoev B et al. 12577067 PLEC1 EGFR Invitro Blagoev B et al. 12577067 EGFR SNX4 In vivo Haft CR et al. 9819414EGFR PRKAR1A In vivo Tortora G et al. 9050991 AMH EGFR In vitro MaggardMA et al. 8596488 EGFR MAP4K1 In vivo Anafi M et al. 9346925 PLCG1 EGFRIn vivo Bedrin MS et al. 9207933 SNX1 EGFR In vivo Haft CR et al.9819414 SNX1 EGFR In vivo Kurten RC et al. 8638121 SNX1 EGFR Two- HaftCR et al. 9819414 hybrid SNX1 EGFR Two- Kurten RC et al. 8638121 hybridEGFR STAT5B In vivo Runge DM et al. 10558875 DEGS1 EGFR In vivo CadenaDL et al. 9188692 DEGS1 EGFR In vitro Cadena DL et al. 9188692 DEGS1EGFR Two- Cadena DL et al. 9188692 hybrid EGFR PIK3C2B In vitro WheelerM et al. 11533253 KRT17 EGFR In vivo Blagoev B et al. 12577067 KRT17EGFR In vitro Blagoev B et al. 12577067 RGS16 EGFR In vivo Derrien A etal. 11602604 HD EGFR In vivo Liu YF et al. 9079622 PITPNA EGFR In vivoKauffmann-Zeh A 7761838 et al. PITPNA EGFR In vitro Kauffmann-Zeh A7761838 et al. EGFR CEBPB In vivo Harmon AW et al. 12095417 EGFR CEBPBIn vitro Harmon AW et al. 12095417 MUC1 EGFR In vivo Li Y et al.11483589 MUC1 EGFR In vivo Schroeder JA et al. 11278868 NCK2 EGFR Invivo Chen M et al. 9737977 NCK2 EGFR In vivo Li W et al. 1333047 EGFRPTK6 In vivo Kamalati T et al. 8940083 EGFR CAV3 In vivo Couet J et al.9374534 EGFR CAV3 In vitro Couet J et al. 9374534 EGFR CRK In vivoHashimoto Y et al. 9642287 EGFR CRK In vitro Hashimoto Y et al. 9642287EGFR GRB7 In vivo Tanaka S et al. 9710451 EGFR GRB7 In vitro Tanaka S etal. 9710451 EGFR SNRPD2 In vivo Blagoev B et al. 12577067 EGFR SNRPD2 Invitro Blagoev B et al. 12577067 EGFR SRC In vitro Sato K et al. 10971656CBLB EGFR In vivo Ettenberg SA et al. 10086340 CBLB EGFR In vitroEttenberg SA et al. 10086340 EGFR CBLC In vivo Keane MM et al. 10362357EGFR CBLC In vitro Keane MM et al. 10362357 EGFR SHC1 In vitro Blagoev Bet al. 12577067 EGFR PTPN6 In vivo Keilhack H et al. 9733788 EGFR PTPN6In vitro Keilhack H et al. 9733788 EPS8 EGFR In vivo Castagnino P et al.7532293 EPS8 EGFR In vivo Di Fiore PP et al. 12127568 SNX2 EGFR In vivoHaft CR et al. 9819414 SNX2 EGFR In vitro Haft CR et al. 9819414 TNCEGFR In vivo Swindle CS et al. 11470832 TNC EGFR In vitro Swindle CS etal. 11470832 EGFR CAMLG In vivo Tran DD et al. 12919676 EPPK1 EGFR Invivo Blagoev B et al. 12577067 EPPK1 EGFR In vitro Blagoev B et al.12577067 EGFR NCK1 In vitro Tang J et al. 9362449 KRT8 EGFR In vivoBlagoev B et al. 12577067 KRT8 EGFR In vitro Blagoev B et al. 12577067EGFR TJP1 In vivo Kaihara T et al. 12708492 VAV1 EGFR In vivo Moores SLet al. 10938113 EGFR STAT5A In vivo Olayioye MA et al. 10358079 EGFRGAB1 In vivo Kameda H et al. 11432805 EGFR TNK2 In vivo Manser E et al.8497321 EGFR TNK2 In vivo Satoh T et al. 8647288 EGFR VAV2 In vivoPandey A et al. 10618391 EGFR VAV2 In vivo Moores SL et al. 10938113EGFR VAV2 In vitro Pandey A et al. 10618391 EGFR VAV2 In vitro Moores SLet al. 10938113 SOS1 EGFR In vivo Qian X et al. 9447973 EGFR MAP3K14 Invivo Habib AA et al. 11116146 EGFR ESR1 In vitro Marquez DC et al.11887937 EGFR ERBB2 In vivo Brockhoff G et al. 11500850 EGFR ERBB2 Invitro Brockhoff G et al. 11500850 SH2D3A EGFR In vivo Lu Y et al.10187783 CDH1 EGFR In vivo Pece S et al. 10969083 EGFR PTPN1 In vivoZhang ZY et al. 8621392 EGFR PTPN1 In vivo Jia Z et al. 7540771 EGFRPTPN1 In vivo Sarmiento M et al. 10889023 EGFR PTPN1 In vivo Li S et al.12573287 EGFR PTPN1 In vitro Zhang ZY et al. 8621392 EGFR PTPN1 In vitroJia Z et al. 7540771 EGFR PTPN1 In vitro Sarmiento M et al. 10889023EGFR PTPN1 In vitro Li S et al. 12573287 VAV3 EGFR In vivo Zeng L et al.11094073 INPPL1 EGFR In vitro Pesesse X et al. 11349134 INPPL1 EGFR Invivo Pesesse X et al. 11349134 EPS15 EGFR In vivo van Delft S et al.9049247 EGFR ARF4 In vitro Kim SW et al. 12446727 EGFR ARF4 In vivo KimSW et al. 12446727 EGFR ARF4 Two- Kim SW et al. 12446727 hybrid EGFRGNAI2 In vivo Zhang BH et al. 11286993 EGFR PDGFRB In vivo Habib AA etal. 9506992 SHC3 EGFR In vivo Nakamura T et al. 9507002 EGFR BTC In vivoMixan B et al. 9528863 EGFR SOS2 In vivo Qian X et al. 10675333 NRG1EGFR In vitro Pinkas-Kramarski R 8702572 et al. EGFR CASP1 In vivo BaeSS et al. 11226410 EGFR CASP1 In vitro Bae SS et al. 11226410 EGFR EREGIn vivo Komurasaki T et al. 9419975 EGFR PTPN11 In vitro Tomic S et al.7673163 EGFR PTPN11 In vivo Tomic S et al. 7673163 PTPRJ EGFR In vivoJallal B et al. 9115287 EGFR RIPK1 In vivo Habib AA et al. 11116146 FEREGFR In vivo Kim L et al. 7623846

Protein-protein interactions typically influence the activity of one orboth interacting partners. For example, a protein-protein interactionmay result in the negative regulation (e.g., inhibition) of one or bothpartners, or may result in the positive regulation (e.g. activation) ofone or both partners. Other functional outcomes also are possible.Exemplary positive and negative regulators for specific RTKs areprovided in Table 3. Exemplary negative regulators of EGFR that formprotein-protein interactions with EGFR include, for instance, SOCS1,SOCS3, SOCS5, and C-CBL. Exemplary positive regulators of EGFR that formprotein-protein interactions with EGFR include, for instance, STAT1,STAT5B, GRB7, HER2, and MUC1.

Methods useful for detection of a protein-protein interaction using adiagnostic specific binding reagent (e.g., interface-specific bindingmolecule) identified using the methods provided herein (such as adiagnostic specific binding reagent, for example an antibody, antibodyfragment, recombinant antibody, scaffold polypeptide with antibodybinding sites, and/or an aptamer) are well known in the art. In someexamples, a fixed biological sample is contacted with a diagnosticspecific binding molecule under conditions that permit (or would permitif it was accessible) binding of the diagnostic specific bindingmolecule to its epitope in the interface between the interactingproteins. Optionally, a control reaction is performed (e.g.,simultaneous with, prior to, or following) to ensure that the conditionsare suitable for the detection reaction to occur. For example, thebiological sample (or a serial section or a parallel-prepared cellsample) also may be contacted with a control antigen-binding molecule(such as, an antibody, antibody fragment, recombinant antibody, scaffoldpolypeptide with antibody binding sites, and/or an aptamer). The controlantigen-binding molecule specifically binds to a non-interactingcomponent of the sample (i) that is not involved in the protein-proteincomplex of interest and (ii) the epitope of which is known to be presentand detectable in the sample under the particular detection conditions.

In exemplary methods, a diagnostic specific binding molecule and anoptional control antigen-binding molecule are antibodies (e.g.,monoclonal antibodies) or antibody fragments. Detection of suchantibodies or antibody fragments is performed by immunostaining, such asillustrated in FIG. 1, which is a standard technique in the art.Detection may by direct or indirect. With direct detection, primaryantibodies (i.e., the antibodies that specifically bind to biologicalcomponent(s) of interest in the sample) are directly labeled, forinstance, with a detectable moiety or with an enzyme that catalyzes areaction leading to a detectable product. With indirect detection, oneor more secondary reagents (such as secondary, tertiary, etc.antibodies) are used to detect the primary antibody (and, as applicable,secondary or subsequent antibodies) and the last of such reagents isdetectable, for instance by labeling with detectable moiety or with anenzyme that catalyzes a reaction leading to a detectable product.Representative immunostaining procedures are provided in the Examples.

Some disclosed methods involve dual detection of an extracellular domain(ED) epitope present on an RTK and an ID (or regulatory domain) epitopeof the RTK. Some RTK ID or regulatory domains have multiple bindingsites for regulatory proteins (e.g., the EGFR regulatory domain, seeFIG. 2). Accordingly, epitopes in the RTK ID may not be accessible infixed biological samples where, at the time of fixation, the RTK wasinvolved in a protein-protein interaction with one or more of itsregulatory proteins (see Tables 3 and 5). In comparison, the ED of RTKsfunction primarily as a ligand-binding domain, and, typically, it is notmasked from antibodies specific for ED epitopes. Accordingly, in somemethods, an antigen-binding molecule specific for an RTK ED (e.g., ananti-RTK ED antibody or fragment thereof) can serve as a control for anantigen-binding molecule specific for a (potentially masked) epitope inan RTK protein-regulatory protein interface. One caveat of interest isthat some RTKs, such as EGFR, HER2, cKit, cMEt, and Her4, may “shed” itsexternal domain or may be mutant and lack its ED (Pedersen et al., Ann.Oncol., 6:745, 2001). Under those circumstances, an antigen-bindingmolecule specific for the RTK ED (e.g., anti-RTK ED antibody or fragmentthereof) would have no target to bind and, therefore, would not bedetected. This circumstance (and useful information that can be gleanedfrom such circumstance) is discussed in detail elsewhere in thisdisclosure.

Some of the foregoing method embodiments and other method embodiments inthis disclosure involve substantially no specific binding of adiagnostic specific-binding molecule (such as a monoclonal antibody) toits epitope (e.g., which is located in the protein-protein interfacebetween an RTK and its regulatory molecule(s)). Substantially no bindingcan be determined by any method available to those of ordinary skill inthe art. For example, substantially no binding of a diagnosticspecific-binding molecule may be relative to the binding of the samediagnostic specific-binding molecule under substantially the sameconditions in another sample in which the epitope of the diagnosticspecific-binding molecule is known to be accessible. In another example,substantially no binding of a diagnostic specific-binding molecule maymean that the detection means (e.g., detectable label or colorimetricreagent) used to visualize the specific binding of the diagnosticspecific-binding molecule can not be seen under ordinary circumstancesfor such detection, e.g., under a light or fluorescence microscope with4×, 10×, or 40× magnification. In still another example, substantiallyno binding of a diagnostic specific-binding molecule means that thediagnostic specific-binding molecule has less than about 25% (such asless than about 20%, less than about 15%, less than about 10%, less thanabout 5%, or less than about 1%) its binding under control circumstances(e.g., in a tissue or cell sample where its epitope is known to beaccessible).

VIII. Prognostic and Predictive Methods

The present disclosure provides methods than can be used to determinethe activation status of a RTK in a biological sample (such as a samplefrom a subject having or suspected of having a neoplasm, for examplecancer), and thus permit predictions about the subject. For example,using diagnostic specific binding reagents identified using the providedmethods permits determination of the prognosis of the subject (such asthe disease outcome without further therapy, for example the likelihoodof survival after initial diagnosis (such as a 1 year or 5 year survivaltime)), as well as predictions about the likelihood that a subject (suchas a subject with a cancer thought or known to express the target RTK)will respond successfully to a particular RTK therapy, such as an RTKtherapy designed to target an ED or ID of the RTK. Therefore, methodsare provided to detect RTK (e.g., EGFR or HER2 or others shown in Table2) molecular interactions in biological samples (such as a fixedbiological sample) which opens the way to predicting RTK status andimportant corollaries in such samples or in subjects from which suchsamples are collected; for example, in neoplastic tissues and/or cellswhere RTK expression or overexpression is believed to play an importantrole in tumorigenesis (e.g., Arnold et al., Oncologist, 6:602, 2006)and/or in cancer patients.

The disclosed diagnostic methods are applicable to any type of neoplasmor cancer or to a subject with any type of neoplasm or cancer, forinstance RTK-expressing (or -overexpressing) cancers. Exemplaryneoplasms useful in all disclosed methods (including predictive methods)are described elsewhere in this disclosure (e.g., Section VI and theExamples). Particular predictive method embodiments involve lung cancer(e.g., non-small cell lung cancer), ovarian cancer, colorectal cancer,liver cancer, head and neck, prostate, and/or glioblastoma and/orsubjects having any of such cancers.

Examples of staining intensity scores which indicate detectable binding(e.g., a positive binding result) of a diagnostic specific bindingreagent with a protein-protein interface or no (or insignificant)detectable binding of a diagnostic specific binding reagent with aprotein-protein interface (e.g., negative binding result) are providedin the Examples below (e.g., see Example 6).

A. Predicting Aggressiveness of RTK-Positive Neoplasms

Determining the prognosis of a subject having a neoplasm, such as acancer, can be achieved by using the diagnostic specific bindingreagents identified using the disclosed methods. Determining theprognosis of a subject allows for a determination or estimation of thedisease (e.g., cancer) outcome in the absence of additional therapy(e.g., estimated time of survival). For example, detection of a direct(e.g., protein-protein) interaction between an RTK and a negativeregulator of RTK function or a positive regulator or RTK function (e.g.,see Table 3) can be used to predict inhibition of RTK function (if thediagnostic specific binding reagent is one that recognizes an interfacebetween an RTK-negative regulatory protein interaction) or predictenhanced RTK function (if the diagnostic specific binding reagent is onethat recognizes an interface between an RTK-positive regulatory proteininteraction), respectively, in a biological sample.

In one example, the diagnostic specific binding reagent is one thatrecognizes an interface between an RTK protein-negative regulatoryprotein interaction (such as the RTK RD amino acids involved in such aninteraction). In such an example, detection of a direct (e.g.,protein-protein) interaction between an RTK and a negative regulator ofRTK indicates that the RTK protein is being downregulated or inhibited.Such a direct interaction between an RTK and a negative regulator of RTKis indicated by the absence of binding (or significantly reducedbinding) by the diagnostic specific binding reagent to the biologicalsample. This is because when the inhibitor is bound to the RTK in thesample, the diagnostic specific binding reagent is unable tospecifically bind in any substantial amount to the RTK-negativeregulatory protein interface. Inhibition of RTK function has importantconsequences in many cells and tissues. For example, in neoplastic cellsand tissues where RTK overexpression or increased biological activity isbelieved to play a role in tumorigenesis (e.g., Arnold et al.,Oncologist, 6:602, 2006), detection of a direct interaction between RTKand a negative regulator of RTK function (e.g., see Table 3) furtherpredicts that a neoplasm may be less aggressive (e.g., less rapidlygrowing, and/or less likely to metastasize). A better prognosis(independent of therapy) for a subject with such a neoplasm also may bepredicted. However, if there is no significant detection of a direct(e.g., protein-protein) interaction between an RTK and a negativeregulator of RTK, this indicates that the RTK protein is not beingdownregulated or inhibited. An absence of direct interaction between anRTK and a negative regulator of RTK is indicated by the presence ofdetectable binding by the diagnostic specific binding reagent to thebiological sample. This is because when the inhibitor is not bound tothe RTK in the sample, the interface that forms between an RTK and anegative regulator is available for binding to the diagnostic specificbinding reagent, and thus binding of the diagnostic specific bindingreagent to the biological sample can be detected. This then indicatesthat the RTK is not being negatively regulated, and may predict that aneoplasm may be more aggressive (e.g., rapidly growing, and/or morelikely to metastasize). A worse prognosis (independent of therapy) for asubject with such a neoplasm also may be predicted.

In a specific example, the RTK is EGFR, wherein detection of a direct(e.g., protein-protein) interaction between EGFR and a negativeregulator of EGFR function (e.g., a SOCS protein, such as SOCS1 orSOCS3, or SOCS5) predicts inhibition of EGFR function in that biologicalsample, and may further predict that a neoplasm may be less aggressive(e.g., less rapidly growing, and/or less likely to metastasize). Abetter prognosis (independent of therapy) for a subject with such aneoplasm also may be predicted.

In another example, the diagnostic specific binding reagent is one thatrecognizes an interface between an RTK protein-positive regulatoryprotein interaction (such as the RTK RD amino acids involved in such aninteraction). In such an example, detection of a direct (e.g.,protein-protein) interaction between an RTK and a positive regulator ofRTK indicates that the RTK protein is being activated or upregulated(e.g., increased RTK expression or biological activity). Such a directinteraction between an RTK and a positive regulator of RTK is indicatedby the absence of binding (or significantly reduced binding) by thediagnostic specific binding reagent to the biological sample. This isbecause when the positive regulator is bound to the RTK in the sample,the diagnostic specific binding reagent is unable to specifically bindin any substantial amount to the RTK-positive regulatory proteininterface. For the opposite of reasons discussed above, a worseprognosis (independent of therapy) for a subject with such a neoplasmalso may be predicted. However, if there is no significant detection ofa direct (e.g., protein-protein) interaction between an RTK and apositive regulator of RTK, this indicates that the RTK protein is notbeing activated or upregulated. An absence of direct interaction betweenan RTK and a positive regulator of RTK is indicated by the presence ofdetectable binding by the diagnostic specific binding reagent to thebiological sample. This is because when the positive regulatory proteinis not bound to the RTK in the sample, the interface that forms betweenan RTK and a positive regulatory protein is available for binding to thediagnostic specific binding reagent, and thus binding of the diagnosticspecific binding reagent to the biological sample can be detected. Thisthen indicates that the RTK is not being activated or upregulated, andmay predict that a neoplasm may be less aggressive (e.g., less rapidlygrowing, and/or less likely to metastasize). A better prognosis(independent of therapy) for a subject with such a neoplasm also may bepredicted.

In a specific example, the RTK is EGFR, wherein detection of a directinteraction between EGFR and a positive regulator of EGFR function(e.g., STAT1, STAT5B, GRB7, HER2, and/or MUC1) predicts activation ofEGFR function in that biological sample (e.g., neoplastic tissue orcells). For the opposite of reasons discussed above, a worse prognosis(independent of therapy) for a subject with such a neoplasm also may bepredicted.

A less-aggressive tumor can be characterized by any parameters known inthe art, including, for instance, decreased growth rate (e.g., increasedrate of apoptosis and/or decreased rate of cell division), decreasedrate of metastasis, and/or increased sensitivity to chemotherapy.

Prognosis for a subject can be characterized by any parameter known inthe art, including, for instance, actual survival after initialdiagnosis (such as 1-year survival, 2-year survival, or 5-yearsurvival), and/or actual survival relative to the average survival forsimilarly situated patients. A better (or good) prognosis entails, e.g.,survival of a patient for more than 1 year after initial diagnosis (suchas more than 2 years or more than 5 years), or survival of a patient formore than 6 months longer (e.g., more than 1 year longer, more than 2years longer, more than 5 years longer) than the average survival forsimilarly situated. A worse (or bad) prognosis entails, e.g., survivalof a patient for less than 5 years after initial diagnosis (such as lessthan 2 years or less than 1 years), or survival of patient less than theaverage survival for similarly situated patients (such as, about 3months less than average survive, about 6 months less than averagesurvive, or about 1 year less than average survival).

Exemplary prognoses based on detecting an interaction (or lack ofinteraction) between an RTK and, e.g., a positive or negative regulatorthat binds the RTK ID (such as those listed in Table 3) are shownschematically in FIGS. 3A and 3B.

B. Predicting Responsiveness of a Cancer Patient to RTK-InhibitorTherapy

Methods of detecting an interaction (or lack of interaction) between atarget RTK and its regulator(s) (such as those shown in Table 3) enablesa variety of predictions with respect to the outcome of RTK inhibitortherapy in a cancer patient. RTK inhibitor therapies include at leasttwo drug classes: (1) RTK antibody therapies, which are typically aredirected to the RTK ED and block binding of an RTK ligand (such as EGFfor the EGFR) to the receptor; thereby, inhibiting RTK activation, and(2) tyrosine kinase inhibitors (TKIs), which inhibit the intracellularkinase domain of a RTK, which also inhibits RTK activation. Exemplaryinhibitory therapies for exemplary target RTKs are provided in Table 8.

TABLE 8 Exemplary RTK inhibitory therapies RTK Antibody Therapy TyrosineKinase Inhibitor EGFR cetuximab (Erbitux ™), gefitinib (Iressa ™),erlotinib panitumumab (Vectibix ™), (Tarceva ™), lapatinib ditosylateIMC-11F8 (Imclone), (GlaxoSmithKline), HKI-272 (Wyeth), matuzumab(Merck_KGA), AEE788 (Novartis), vandetanib zalutumumab, (Zactima ™;Astrazeneca), XL647 (Exelixis), BMS-599626 (Bristol-Myers Squibb), BIBW2992 (Boehringer Ingelheim), ARRY-543, MP-412, JNJ- 26483327, PF-299804,Neratinib FGFR1 FGFR2 FGFR3 Sunitinib, CHIR258 FGFR4 Flt3 ENMD-981693,KW-2449, ABT-869, AC220, SU014813, CHIR258 HER2 (ErbB2) Trastuzumab(Herceptin ®), Lapatinib (Tykerb ®), XL647, ARRY- bevacizumab(Avastin ®), 543, BMS-599626, MP-412, BIBW pertuzumab (OmniTarg ™),2992, JNJ-26483327, PF-299804, ZM105180 (Zemab ®), ARRY-380, Neratinib,INSM-18 ertumaxomab (Rexonum) HER3 (ErbB3) PF-299804 HER4 (ErbB4)PF-299804 HGFR (MET) AMG 102 (Amgen), XL880, XL184, PF-02341066, SGX523,MetMab (Genentech) JNJ-38877605, ARQ197, PHA665752, MP470, ARQ-650RP,MGCD265, MK- 2461, PHA665752 IGF-1R AMG 479 (Amgen), CP- OSI-906, XL228,BMS-754807, 751,871, R1507, RAV12, AMG479, INSM-18 IMC-A12 (ImClone),BIIB022 Kit Imatinib (Gleevec ™), OSI-930, OSI- 817, sunitinib, XL820,motesanib diphosphate (Amgen), ENMD-981693, KW-2449, MP470, ABT-869,AC220, Telatinib, SU014813, Cediranib, CHIR258, MP470, STI571, DasatinibPDGFR CP-547,632, sunitinib, motesanib diphosphate (Amgen), ABT-869,AC220, Telatinib, SU014813, CHIR258, XL820, Dasatinib Ret XL184, MP470,AC220, Vandetanib (Zactima ™) RON MGCD265 VEGFR1 Sunitinib, OSI-930,OSI-817, XL647, XL820, XL880, XL184, motesanib diphosphate (Amgen),cediranib, CHIR258, MGCD265 VEGFR2 CP-868,596, sunitinib, XL184, XL820,(KDR/flk-1) XL880, vandetanib (Zactima ™), motesanib diphosphate(Amgen), ENMD-981693, Telatinib, SU014813, MGCD265 VEGFR3 (Flt4)Sunitinib, XL228, motesanib diphosphate (Amgen), JNJ-26483327,Telatinib, MGCD265

Some method embodiments involve one or both of the foregoing classes ofRTK inhibitors, and thus can be used to predict the response of a cancerpatient to any of the inhibitors listed in Table 7. Particular methodembodiments involve predicting the response of cancer patients to thefollowing EGFR inhibitors: cetuximab (Erbitux™), panitumumab(Vectibix™), gefitinib (Iressa™), or erlotinib (Tarceva™), or anycombination thereof (such as, cetuximab (Erbitux™) or panitumumab(Vectibix™), gefitinib (Iressa™) or erlotinib (Tarceva™), or cetuximab(Erbitux™), panitumumab (Vectibix™), gefitinib (Iressa™) or erlotinib(Tarceva™)), or the HER2 inhibitors Herceptin® and Avastin®.

Exemplary predictions based on detecting an interaction (or lack ofinteraction) between an RTK and, e.g., a positive or negative regulatorthat binds the RTK ID (such as those listed in Table 3) are shownschematically in FIG. 3A (negative regulator) and 3B (positiveregulator). Exemplary predictions based on detecting an interaction (orlack of interaction) between an RTK and a negative regulator that bindsthe RTK ID (such as those listed in Table 3) are shown in Table 9.

TABLE 9 Exemplary Predicted Therapeutic Response Predicted ID-BindingED-Binding Therapeutic Molecule Molecule RTK Implication TherapeuticResponse 1 Positive Positive RTK is present ED-based therapy SensitiveRDIM (e.g. SOCS3) (e.g., EGFR Ab) is absent ID-based therapy Sensitive(e.g., TKI) 2 Negative Positive RTK is present ED-based therapyResistant RDIM (e.g. SOCS3) (e.g., EGFR Ab) is present ID-based therapyResistant (e.g., TKI) 3 Positive Negative Mutant/Cleaved RTK ED-basedtherapy Resistant is present (e.g., EGFR Ab) RDIM (e.g. SOCS3) ID-basedtherapy Sensitive is absent (e.g., TKI) 4 Negative Negative RTK isabsent ED-based therapy No Response (e.g., EGFR Ab) ID-based therapy NoResponse (e.g., TKI) ID = RTK intracellular (or regulatory) domain; ED =RTK extracellular domain; RDIM = regulatory domain inhibitory molecule;Ab = antibody (e.g., monoclonal or otherwise engineered antibody)

In one method embodiment, an interaction between the ID (e.g.,regulatory domain) of RTK and at least one of its negative regulators(e.g., see Table 3) is detected (e.g., by masking of the epitope of adiagnostic specific binding molecule (such as a monoclonal antibody,including clone 5B7 (see, e.g., Examples)). Optionally, butadvantageously, the presence of full-length (or substantiallyfull-length) RTK also is detected using an antigen-binding molecule(e.g., monoclonal antibody, such as clone 3C6 for EGFR) specific for theRTK ED. In this example, the diagnostic specific binding molecule (e.g.,clone 5B7 for EGFR) is excluded from its binding site and, therefore, isnot detected, while the external-domain antigen-binding molecule (e.g.,clone 3C6 for EGFR) binds to its epitope and is detected (right panel ofFIG. 3A). These circumstances support a prediction that a therapydesigned to inhibit RTK function likely would not be effective or wouldbe less effective than in the absence of the negative regulator. That is(solely for illustration purposes (and not to be limited by mechanism orimplication of a mechanism)): Providing an RTK inhibitor to a subject inwhich RTK function already was inhibited maybe analogous to applying thebrakes in a car that is already at a stop.

In another method embodiment, an interaction between the RTK ID and itsnegative regulator (e.g., see Table 3) is lacking (e.g., as demonstratedby the binding of a diagnostic specific binding molecule (such as amonoclonal antibody, including clone 5B7 (see, e.g., Examples)) to itsepitope on the RTK, which would otherwise be masked by the RTK-negativeregulator interaction). Optionally, but advantageously, the presence ofthe RTK ED (i.e., full-length or substantially full-length RTK) also isdetected using an antigen-binding molecule (e.g., monoclonal antibody,including clone 3C6 for EGFR) specific for that domain. In this example,the diagnostic specific binding molecule (e.g., clone 5B7 for EGFR)specifically binds its epitope in the RTK regulatory domain and,therefore, is detected, and the external-domain antigen-binding molecule(e.g., clone 3C6) also binds to its epitope and is detected (left panelof FIG. 3A). These circumstances support a prediction that a therapydesigned to inhibit RTK function (e.g., see Table 8) likely would beeffective or would be more effective than in the presence of thenegative regulator, as there are fewer “brakes” on oncogenic RTKfunction and the therapy has something to inhibit.

In still another method embodiment, an interaction between the ID of anRTK and its negative regulator (e.g., see Table 3) is lacking (e.g., asdemonstrated by the binding to the RTK of a diagnostic specific bindingmolecule (such as a monoclonal antibody, including clone 5B7 (see, e.g.,Examples)) to its epitope, which would otherwise be masked by theRTK-negative regulator interaction). Optionally, but advantageously, thepresence or absence of the RTK ED (i.e., full-length or substantiallyfull-length RTK) also is detected using an antigen-binding molecule(e.g., monoclonal antibody, including clone 3C6 for EGFR) specific forthat domain. In this example, the diagnostic specific binding molecule(e.g., clone 5B7 for EGFR) specifically binds its epitope in the RTK IDand, therefore, is detected; however, it also is determined that the RTKED is lacking (e.g., a mutant or N-terminal truncated RTK) by failure tobind of an antigen-binding molecule specific for that domain (e.g.,monoclonal antibody, including clone 3C6 for EGFR) (middle panel of FIG.3A). These circumstances support a prediction that an antibody therapydesigned to inhibit RTK function by blocking ligand binding to the RTKexternal domain (e.g., those listed in Table 6) likely would be noteffective because such domain is lacking. On the other hand, thesecircumstances further support a prediction that a TKI therapy (e.g.,those listed in Table 6), which inhibits the tyrosine kinase activitylocalized in the RTK ID, likely would be effective or would be moreeffective than in the presence of the negative regulator.

In one method embodiment, an interaction between the ID (e.g.,regulatory domain) of RTK and at least one of its positive regulators(e.g., see Table 3) is detected (e.g., by masking of the epitope of adiagnostic specific binding molecule. Optionally, but advantageously,the presence of full-length (or substantially full-length) RTK also isdetected using an antigen-binding molecule (e.g., monoclonal antibody,such as clone 3C6 for EGFR) specific for the RTK ED. In this example,the diagnostic specific binding molecule is excluded from its bindingsite and, therefore, is not detected, while the external-domainantigen-binding molecule (e.g., clone 3C6 for EGFR) binds to its epitopeand is detected (right panel of FIG. 3B). These circumstances support aprediction that a therapy designed to inhibit RTK function likely wouldbe effective. That is (solely for illustration purposes (and not to belimited by mechanism or implication of a mechanism)): Providing an RTKinhibitor to a subject in which RTK function is being activated will beeffective as the oncogenic RTK is very activity and thus there issignificant RTK activity to be inhibited.

In another method embodiment, an interaction between the RTK ID and itspositive regulator (e.g., see Table 3) is lacking (e.g., as demonstratedby the binding of a diagnostic specific binding molecule to its epitopeon the RTK, which would otherwise be masked by the RTK-positiveregulator interaction). Optionally, but advantageously, the presence ofthe RTK ED (i.e., full-length or substantially full-length RTK) also isdetected using an antigen-binding molecule (e.g., monoclonal antibody,including clone 3C6 for EGFR) specific for that domain. In this example,the diagnostic specific binding molecule specifically binds its epitopein the RTK regulatory domain and, therefore, is detected, and theexternal-domain antigen-binding molecule (e.g., clone 3C6) also binds toits epitope and is detected (left panel of FIG. 3B). These circumstancessupport a prediction that a therapy designed to inhibit RTK function(e.g., see Table 8) likely would not be effective or would be lesseffective, for example, because the oncogenic RTK is less activity thanif the positive regulatory were present, thus the RTK inhibitory therapyhas less to inhibit.

In still another method embodiment, an interaction between the ID of anRTK and its positive regulator (e.g., see Table 3) is lacking (e.g., asdemonstrated by the binding to the RTK of a diagnostic specific bindingmolecule to its epitope, which would otherwise be masked by theRTK-positive regulator interaction). Optionally, but advantageously, thepresence or absence of the RTK ED (i.e., full-length or substantiallyfull-length RTK) also is detected using an antigen-binding molecule(e.g., monoclonal antibody, including clone 3C6 for EGFR) specific forthat domain. In this example, the diagnostic specific binding molecule(e.g., clone 5B7 for EGFR) specifically binds its epitope in the RTK IDand, therefore, is detected; however, it also is determined that the RTKED is lacking (e.g., a mutant or N-terminal truncated RTK) by failure tobind of an antigen-binding molecule specific for that domain (e.g.,monoclonal antibody, including clone 3C6 for EGFR) (middle panel of FIG.3B). These circumstances support a prediction that an antibody therapydesigned to inhibit RTK function by blocking ligand binding to the RTKexternal domain (e.g., those listed in Table 6) likely would be noteffective because such domain is lacking, and also that therapy designedto inhibit RTK function (e.g., see Table 8) likely would not beeffective or would be less effective, for example, because the oncogenicRTK is less activity than if the positive regulatory protein werepresent, thus the RTK inhibitory therapy has less to inhibit.

The response of a subject to RTK inhibitor therapy can be measured byany relevant parameter known in the art. In some method embodiments, asubject response is cessation or slowing of tumor growth (as measured,for example, by tumor size), decrease in tumor cell proliferation,increase in tumor cell apoptosis, and/or decreased level of relevanttumor marker(s). In other method embodiments, a subject response is atleast a 50% slowing of tumor growth or tumor cell proliferation ascompared to pre-treatment growth (such at least a 40% slowing, at leasta 30% slowing, at least a 20% slowing, or at least a 10% slowing). Inother method embodiments, a subject response is at least a 50% increasein tumor cell apoptosis as compared to pre-treatment levels (such atleast a 40% increase, at least a 30% increase, at least a 20% increase,or at least a 10% increase).

IX. Kits

Any of the diagnostic specific-binding molecules described in thisdisclosure can be supplied in the form of a kit useful, at least, forperforming the methods described herein. In one embodiment of such akit, an appropriate amount of at least one diagnostic specific-bindingmolecule (e.g., monoclonal antibody (such as clone 5B7) or fragmentthereof) is provided in one or more containers. In other embodiments, atleast one diagnostic specific-binding molecule (e.g., monoclonalantibody (such as clone 5B7) or fragment thereof) may be providedsuspended in an aqueous solution or as a freeze-dried or lyophilizedpowder, for instance. The container(s) in which the at least onediagnostic specific-binding molecule (e.g., monoclonal antibody (such asclone 5B7) or fragment thereof) is supplied can be any conventionalcontainer that is capable of holding the supplied form, for instance,microfuge tubes, ampoules, or bottles. The amount of diagnosticspecific-binding molecule (e.g., monoclonal antibody (such as clone 5B7)or fragment thereof) supplied can be any appropriate amount, such asfrom about 1 to about 5 μg/ml.

In other embodiments, control slides upon which are mounted one or moretissue or cell preparations (e.g., xenografts, cell pellets, or clottedcells) that may serve as positive and/or negative controls for adiagnostic specific-binding molecule (e.g., monoclonal antibody (such asclone 5B7) or fragment thereof) may be provided in an appropriate andseparate container. In some instances, A431, DU145, and/or Caski cells(or xenografts prepared therewith) may serve as a positive control. Inother instances, MCF-7 cells (or xenografts prepared therewith) mayserve as a negative control.

Other kit embodiments will include means for detection of the diagnosticspecific-binding molecule, such as secondary antibodies (e.g., goatanti-rabbit antibodies or rabbit anti-mouse antibodies). In some suchinstances, the secondary antibody will be directly labeled with adetectable moiety (as described elsewhere in this disclosure). In otherinstances, the primary or secondary (or higher-order) antibody will beconjugated to a hapten (such as biotin, DNP, and/or FITC), which isdetectable by a detectably labeled cognate hapten-binding molecule(e.g., streptavidin (SA)-horse radish peroxidase, SA-alkalinephosphatase, and/or SA-QDot™). Some kit embodiments may includecolorimetric reagents (e.g., DAB, and/or AEC) in suitable containers tobe used in concert with primary or secondary (or higher-order)antibodies that are labeled with enzymes for the development of suchcolorimetric reagents.

In one embodiment, a kit includes instructional materials disclosingmethods of use of the kit contents (e.g., diagnostic specific-bindingmolecule) in a disclosed method. The instructional materials may bewritten, in an electronic form (e.g. computer diskette or compact disk)or may be visual (e.g. video files). The kits may also includeadditional components to facilitate the particular application for whichthe kit is designed. Thus, for example, the kits may additionallyinclude buffers and other reagents routinely used for the practice of aparticular method. Such kits and appropriate contents are well known tothose of skill in the art.

The following examples are provided to illustrate certain particularfeatures and/or embodiments. These examples should not be construed tolimit the invention to the particular features or embodiments described.

EXAMPLES Example 1 An Exemplary Monoclonal Antibody Specific for EGFRRegulatory Domain

This Example describes an exemplary RD-binding molecule; moreparticularly a monoclonal antibody that binds an epitope in the EGFRinhibitory subdomain. This antibody has the added advantage that it willidentify not only full-length EGFR, but also truncated mutant forms ofEGFR, which have been shown to be constitutively activated (Pedersen etal., Ann. Oncol., 12(6):745-60, 2001). Similar methods can be used toidentify antibodies for other RTK intracellular domains.

A computer program (DNASTAR™, Madison, Wis.) was used for the selectionof immunogenic peptide sequences within the EGFR intracellular domain.The program examined the input protein sequence for short (e.g., lessthan 20 contiguous amino acids) sequences that likely had a highprobability for producing an antibody response in animals immunized withimmunogens including such short sequences.

One identified short sequence was LDNPDYQQDFFPKEAKPNG (L2G Peptide; SEQID NO: 2), which, by computer analysis, had high antigenicity, highhydrophilic regions, and high surface probability regions. This aminoacid sequence was selected and a corresponding peptide was synthesizedusing a commercially available service (Anaspec, San Jose, Calif.).

The synthesized peptide was conjugated to Keyhole Limpet Hemocyanin(KLH) using standard methods. Rabbits were immunized with theKLH-peptide conjugate by a commercially available service (StrategicDiagnostics, Inc. Newark, Del.).

Rabbit sera containing antibodies specific for the L2G Peptide wereidentified by ELISA assay. The animal with the strongest serum titer wasselected for a splenectomy. The viable spleen was shipped to Epitomics,Inc (Burlingame, Calif.) overnight where the immunized spleen cells wereprepared for fusion with an immortalized cell line (240E-w) asdescribed, e.g., in U.S. Pat. No. 5,675,063 or European Pat. No.EP0815213B1.

Hybridoma supernatants were tested by ELISA assay for the presence ofantibodies specific for the L2G Peptide. One hybridoma was selectedbased on a relatively high antibody titer in the correspondingsupernatant. The specificity of antibodies produced by the selectedhybridoma cell line was confirmed by immunohistochemistry (IHC) testingon known EGFR-positive tissues (including squamous cell carcinoma of thelung, colon adenocarcinomas, and normal skin) The hybridoma cell linedelivered by the manufacturer was subcloned to homogeneity to isolate ahigh-producing hybridoma clone designated 5B7.

A Western blot analysis was performed to ensure the specificity of clone5B7. Total protein lysates were prepared from A431 cells, which areknown to express high levels of EGFR on their cell surface, and fromBT474 cells, which are negative for EGFR, but which express related EGFRfamily members, EGFR2 and EGFR3. As shown in FIG. 4, clone 5B7 and amouse monoclonal antibody specific for the EGFR external domain (clone3C6) recognized the same 170 kDa band, which is consistent with the sizeof the EGFR protein. The lack of staining of the BT474 cell lysates forboth antibodies indicated that neither antibody cross reacted with EGFRfamily members that have conserved homology. Thus, the 3C6 and 5B7antibodies were specific for EGFR.

Example 2 Exemplary Methods for Immunohistochemical Staining of Tissuewith EGFR-Specific Antibodies

Immunohistochemistry is the well-known method and variations on suchmethods are readily determined with routine experimentation by those ofordinary skill in the art (see, e.g., Dabbs, DiagnosticImmunohistochemistry, Churchill Livingstone, 2002). Exemplary methodsfor detecting in FFPE tissue by manual IHC an EGFR RD-binding molecule(e.g., monoclonal antibody clone 5B7) or an antigen-binding moleculespecific for the EGFR extracellular domain (e.g., monoclonal antibodyclone 3C6) are provided in Table 10. One skilled in the art willrecognize that similar methods provide in the Tables below can be usedfor diagnostic specific binding reagents for other RTKs.

TABLE 10 Exemplary IHC Methods Step # Manual IHC Assay 0 Fresh tissue isplaced in a fixative (such as, 10% neutral buffered formalin) forapproximately 12-48 hours at room temperature. Then, the tissue isdehydrated through graded alcohols (e.g., 50% to 70% to 90% to 95% to100% EtOH) for 1-2 hours at each grade, and infiltrated with a clearingreagent (such as, xylene) for 3-5 hours at room temperature. The clearedtissue is placed in melted (approximately 63 degrees C) paraffin for 3-6hours. Samples are removed and embedded in paraffin blocks forsubsequent microtome sectioning. 3-10 μm sections are cut and placed onglass slides. 1 Deparaffinize tissue sections in xylene; then, rehydratethrough graded alcohols to distilled water. 2 Place tissue sections in0.5% v/v hydrogen peroxide/methanol for approximately 10 minutes. 3Pretreat slides for antigen retrieval using an appropriate method (e.g.,high-temperature antigen unmasking, trypsin, etc.) if required. 4 Washslides with distilled water for approximately 5 minutes. 5 Wash slidesin saline buffer (e.g., PBS, TBS) for 5 minutes. 6 Cover tissue sectionswith blocking reagent (e.g., 10% v/v normal rabbit serum in buffer) forapproximately 10 minutes. 7 Remove excess blocking reagent and replacewith primary antibody (e.g., rabbit monoclonal antibody or mousemonoclonal antibody) diluted in blocking reagent as required forapproximately 60 minutes at 25° C. or overnight at 4° C. 8 Wash twice inbuffer for approximately 5 minutes per wash. 9 Remove excess buffer andincubate tissue sections with biotinylated secondary antibody (e.g.,biotinylated rabbit anti-mouse antibody or biotinylated goat anti-rabbitantibody as appropriate for the subject primary antibody) diluted inblocking reagent for 30 minutes at 25° C. 10 Wash twice in buffer forapproximately 5 minutes per wash. 11 Remove excess buffer and incubatetissue sections with streptavidin-horse radish peroxidase (HRP)conjugate for 30 minutes at 25° C. 12 Wash twice in buffer forapproximately 5 minutes per wash. 13 Develop detectable color with3,3′-diaminobenzidine tetrahydrochloride (DAB) at room temperature forapproximately 5-10 minutes. 14 Rinse slides in water. 15 If desiredcounterstain with hematoxylin (e.g., Carson, Histotechnology: A Self-Instructional Text, Chicago:ASCP Press, 1997). 16 Dehydrate, clear andmount coverslip on slides.

IHC for the detection antibodies specific for the EGFR regulatory domainalso can be performed on automated staining platforms, such as theBenchMark™ series instruments manufactured by Ventana Medical Systems(Tucson, Ariz.). An exemplary assay for the detection of a monoclonalantibody specific for the EGFR regulatory domain (e.g., clone 5B7) on aBenchMark™ series automated tissue stainer is described in Table 11.

TABLE 11 Exemplary method of monoclonal antibody detection Step #Anti-EGFR Regulatory Domain Antibody Staining (Automated Assay) 1 ***Select EZ Prep *** 2 *** Start Timed Steps *** 3 *** Mixers Off *** 4Warmup Slide to 75° C., and Incubate for 4 Minutes 5 Apply EZPrep VolumeAdjust 6 Rinse Slide 7 Apply EZPrep Volume Adjust 8 Rinse Slide 9 ApplyEZPrep Volume Adjust 10 Apply Coverslip 11 Warmup Slide to 76° C., andIncubate for 4 Minutes 12 Rinse Slide 13 Apply Depar Volume Adjust 14Apply Coverslip 15 Disable Slide Heater 16 *** Mixers On *** 17 [Short -8 Minute Conditioning] 18 Rinse Slide 19 Apply Long Cell Conditioner #120 Apply CC Coverslip Long 21 *** Select SSC Wash *** 22 Warmup Slide to95° C., and Incubate for 8 Minutes 23 [Mild - 30 Minute Conditioning] 24Apply Cell Conditioner #1 25 Apply CC Medium Coverslip No BB 26 WarmupSlide to 100° C., and Incubate for 4 Minutes 27 Apply CC MediumCoverslip No BB 28 Apply Cell Conditioner #1 29 Apply CC MediumCoverslip No BB 30 Apply Cell Conditioner #1 31 Apply CC MediumCoverslip No BB 32 Apply Cell Conditioner #1 33 Apply CC MediumCoverslip No BB 34 Apply Cell Conditioner #1 35 Apply CC MediumCoverslip No BB 36 Apply Cell Conditioner #1 37 Apply CC MediumCoverslip No BB 38 [Standard - 60 Minute Conditioning] 39 Apply CellConditioner #1 40 Apply CC Medium Coverslip No BB 41 Apply CellConditioner #1 42 Apply CC Medium Coverslip No BB 43 Apply CellConditioner #1 44 Apply CC Medium Coverslip No BB 45 Apply CellConditioner #1 46 Apply CC Medium Coverslip No BB 47 Apply CellConditioner #1 48 Apply CC Medium Coverslip No BB 49 Apply Short CellConditioner #1 50 Apply CC Medium Coverslip No BB 51 Apply CellConditioner #1 52 Apply CC Medium Coverslip No BB 53 Disable SlideHeater 54 Incubate for 8 Minutes 55 Rinse Slide With Reaction Buffer 56Adjust Slide Volume With Reaction Buffer 57 Apply Coverslip 58 RinseSlide With Reaction Buffer 59 Adjust Slide Volume With Reaction Buffer60 Apply Coverslip 61 *** Procedure Synchronization *** 62 Warmup Slideto 37° C., and Incubate for 4 Minutes 63 Rinse Slide With ReactionBuffer 64 Adjust Slide Volume With Reaction Buffer 65 Apply One Drop ofI-VIEW INHIBITOR, Apply Coverslip, and Incubate for 4 Minutes 66 RinseSlide With Reaction Buffer 67 Adjust Slide Volume With Reaction Buffer68 Apply Coverslip 69 Warmup Slide to 37° C., and Incubate for 4 Minutes70 Rinse Slide With Reaction Buffer 71 Adjust Slide Volume With ReactionBuffer 72 Apply Coverslip 73 Apply One Drop of Antibody (e.g., clone5B7), and Incubate for [0 Hr 16 Min] 74 Rinse Slide With Reaction Buffer75 Adjust Slide Volume With Reaction Buffer 76 Apply Coverslip 77 WarmupSlide to 37° C., and Incubate for 4 Minutes 78 Rinse Slide With ReactionBuffer 79 Adjust Slide Volume With Reaction Buffer 80 Apply One Drop ofI-VIEW BIOTIN Ig, Apply Coverslip, and Incubate for 8 Minutes 81 RinseSlide With Reaction Buffer 82 Adjust Slide Volume With Reaction Buffer83 Apply One Drop of I-VIEW SA-HRP, Apply Coverslip, and Incubate for 8Minutes 84 Rinse Slide With Reaction Buffer 85 Adjust Slide Volume WithReaction Buffer 86 Apply Coverslip 87 Rinse Slide With Reaction Buffer88 Adjust Slide Volume With Reaction Buffer 89 Apply One Drop of I-VIEWDAB and One Drop of I-VIEW H₂O₂, Apply Coverslip, Incubate for 8 Minutes90 Rinse Slide With Reaction Buffer 91 Adjust Slide Volume With ReactionBuffer 92 Apply One Drop of I-VIEW COPPER, Apply Coverslip, and Incubatefor 4 Minutes 93 Rinse Slide With Reaction Buffer 94 Adjust Slide VolumeWith Reaction Buffer 95 Apply One Drop of [HEMATOXYLIN II](Counterstain), Apply Coverslip, and Incubate for [4 Minutes] 96 RinseSlide With Reaction Buffer 97 Adjust Slide Volume With Reaction Buffer98 Apply Coverslip 99 Rinse Slide With Reaction Buffer 100 Adjust SlideVolume With Reaction Buffer 101 Apply One Drop of [BLUING REAGENT] (PostCounterstain), Apply Coverslip, and Incubate for [4 Minutes] 102 RinseSlide With Reaction Buffer 103 Apply Coverslip 104 Disable Slide Heater105 *** Select Optional Wash *** 106 *** Select SSC Wash *** 107 ***Start Timed Steps *** 108 Rinse Slide With Reaction Buffer

An exemplary assay for the detection of a monoclonal antibody specificfor the EGFR external domain (e.g., clone 3C6) on a BenchMark™ seriesautomated tissue stainer is described in Table 12.

TABLE 12 Exemplary method for detecting RTK extracellular domain Step #Anti-EGFR External Domain Antibody Staining (Automated Assay) 1 ***Select EZ Prep *** 2 *** Start Timed Steps *** 3 *** Mixers Off *** 4Warm Slide to 75° C., and Incubate for 4 Minutes 5 Apply EZPrep VolumeAdjust 6 Rinse Slide 7 Apply EZPrep Volume Adjust 8 Rinse Slide 9 ApplyEZPrep Volume Adjust 10 Apply Coverslip 11 Warm Slide to 76° C., andIncubate for 4 Minutes 12 Rinse Slide 13 Apply Depar Volume Adjust 14Apply Coverslip 15 Disable Slide Heater 16 *** Mixers On *** 17 DisableSlide Heater 18 *** Select SSC Wash *** 19 Rinse Slide With ReactionBuffer 20 Adjust Slide Volume With Reaction Buffer 21 Apply Coverslip 22Rinse Slide With Reaction Buffer 23 Adjust Slide Volume With ReactionBuffer 24 Apply Coverslip 25 *** Procedure Synchronization *** 26 WarmSlide to 37° C., and Incubate for 4 Minutes 27 Rinse Slide With ReactionBuffer 28 Adjust Slide Volume With Reaction Buffer 29 Apply One Drop ofI-VIEW INHIBITOR, Apply Coverslip, and Incubate for 4 Minutes 30 RinseSlide With Reaction Buffer 31 Adjust Slide Volume With Reaction Buffer32 Apply One Drop of [PROTEASE 1] (Enzyme), Apply Coverslip, andIncubate for [8 Minutes] 33 Rinse Slide With Reaction Buffer 34 AdjustSlide Volume With Reaction Buffer 35 Apply Coverslip 36 Warm Slide to37° C., and Incubate for 4 Minutes 37 Rinse Slide With Reaction Buffer38 Adjust Slide Volume With Reaction Buffer 39 Apply Coverslip 40 ApplyOne Drop of Antibody (e.g., clone 3C6), and Incubate for [0 Hr 32 Min]41 Rinse Slide With Reaction Buffer 42 Adjust Slide Volume With ReactionBuffer 43 Apply Coverslip 44 Warm Slide to 37° C., and Incubate for 4Minutes 45 Rinse Slide With Reaction Buffer 46 Adjust Slide Volume WithReaction Buffer 47 Apply One Drop of I-VIEW BIOTIN Ig, Apply Coverslip,and Incubate for 8 Minutes 48 Rinse Slide With Reaction Buffer 49 AdjustSlide Volume With Reaction Buffer 50 Apply One Drop of I-VIEW SA-HRP,Apply Coverslip, and Incubate for 8 Minutes 51 Rinse Slide With ReactionBuffer 52 Adjust Slide Volume With Reaction Buffer 53 Apply Coverslip 54Rinse Slide With Reaction Buffer 55 Adjust Slide Volume With ReactionBuffer 56 Apply One Drop of I-VIEW DAB and One Drop of I-VIEW H₂O₂,Apply Coverslip, Incubate for 8 Minutes 57 Rinse Slide With ReactionBuffer 58 Adjust Slide Volume With Reaction Buffer 59 Apply One Drop ofI-VIEW COPPER, Apply Coverslip, and Incubate for 4 Minutes 60 RinseSlide With Reaction Buffer 61 Adjust Slide Volume With Reaction Buffer62 Apply One Drop of [HEMATOXYLIN II] (Counterstain), Apply Coverslip,and Incubate for [4 Minutes] 63 Rinse Slide With Reaction Buffer 64Adjust Slide Volume With Reaction Buffer 65 Apply Coverslip 66 RinseSlide With Reaction Buffer 67 Adjust Slide Volume With Reaction Buffer68 Apply One Drop of [BLUING REAGENT] (Post Counterstain), ApplyCoverslip, and Incubate for [4 Minutes] 69 Rinse Slide With ReactionBuffer 70 Apply Coverslip 71 Disable Slide Heater 72 *** Select OptionalWash *** 73 *** Select SSC Wash *** 74 *** Start Timed Steps *** 75Rinse Slide With Reaction Buffer

Example 3 Antibody Specific for EGFR Regulatory Domain EpitopeUnexpectedly does not Substantially Bind to Some EGFR-Positive Tissues

This Example demonstrates that RD-binding molecules, such as clone 5B7,exhibited differential binding to EGFR-positive tissues (as detected byan antibody specific for the EGFR external domain). As described in moredetail below, but without being limited to a single theory, thisdifferential binding is believed to be due to the differentialexpression of EGFR regulatory proteins (e.g., SOCS proteins like SOCS1or SOCS3) in EGFR-positive tissues. Such regulatory proteins, whendirectly associated with the EGFR regulatory domain, mask the epitopesof RD-binding molecules.

A. Normal Human Tissues

The staining by IHC of antibodies specific for the EGFR regulatorydomain (i.e., clone 5B7) and external domain (i.e., clone 3C6) in FFPE30 normal human tissues were compared. Tissue arrays were obtained fromUSBiomax (Igamsville, Md.; Cat. No. FDA801). Automated stainingprotocols as described in Example 2 were used to stain the tissue arrayson a BenchMark™ automated tissues stainer.

As shown in FIG. 5, extracellular-domain-specific clone 3C6 positivelystained skin, testis, tonsil, liver and placenta tissues, which isconsistent with known EGFR expression patterns in normal tissue. FIG. 5also shows that regulatory-domain-specific clone 5B7 reacted positivelywith normal skin, testis, and tonsil. However, surprisingly, thisantibody did not react with normal liver (see, e.g., FIG. 5B) and hadvariable reactivity with placental EGFR depending on the stage ofdevelopment of the placenta (Virchows Archiv A Pathol Anat., 420:385393, 1992).

B. Human NSCLC Tumors

Non-small cell lung cancer (NSCLC) cells are known to express EGFR inapproximately 75% of tumors. Regulatory-domain-specific clone 5B7 andexternal-domain-specific clone 3C6 were used to stain a cohort of NSCLCcases from three commercially available tissue micro arrays (Array LC801and Array LC819 (Biomax; Ijamsville, Md.) and Array IMH-305 (Imgenex(San Diego, Calif.)).

As shown in Table 13, Subpart A, clone 3C6 detected EGFR in 83% of thelung cases (as would be expected based on literature estimates of EGFRstaining in NSCLC) while clone 5B7 stained positively 65% of lungtumors. This corresponds to an 18.5% discordance between clone 3C6 andclone 5B7 with the latter exhibiting no staining in 38 cases that werepositive for clone 3C6 staining

TABLE 13 Summary of NSCLC Immunohistochemistry Study 3C6 5B7 PositiveNegative Total Subpart A Positive 132 1 133 Negative 38 34 72 Total 17035 205 Subpart B Sensitivity 78% Specificity 97% Overall 81% Kappa 67%

As summarized in Table 13, Subpart B, the two antibodies each stainedpositively in 78% of cases (i.e., Sensitivity (132/[132+38]); the twoantibodies each stained negatively in 97% of cases (i.e., Specificity(34/[1+34]). The overall agreement was 81% ([132+43]/205). The Kappastatistic, which is another measure of agreement, can be interpreted asfollows: <0=No agreement, 0.0-0.19=Poor agreement, 0.20-0.39=Fairagreement, 0.40-0.59=Moderate agreement, 0.60-0.79=Substantial agreementand 0.80-1.00=Almost perfect agreement (Landis and Koch, Biometrics,33:159-174, 1977). The Kappa score for 3C6 versus 5B7 was 67% whichfalls into the substantial agreement category.

Particular examples demonstrating differences in the binding of EGFRregulatory-domain-specific clone 5B7 and EGFR external-domain-specificclone 3C6 to squamous cell carcinomas of the lung are shown in FIG. 6.Case 1 (left panel) showed equivalent staining of cells by the twoantibodies, which indicates (among other things) that there is not adifference in the general sensitivity of clone 5B7 as compared to clone3C6 when the epitope for each is accessible. Case 2 (right panel) showeddistinctly different staining between the two antibodies with clone 5B7being negative and clone 3C6 being 3+ positive (0-3+ scale).

The differential binding of clone 5B7 (specific for the EGFRintracellular regulatory domain) as compared to clone 3C6 (specific forthe EGFR extracellular domain) in normal and neoplastic tissues as shownin this Example strongly supports the belief that the clone 5B7 epitopewas accessible only in some tissues.

Example 4 Epitope Mapping of Monoclonal Antibody Clone 5B7

The epitope for the EGFR regulatory-domain-specific monoclonal antibody,clone 5B7, was mapped by peptide inhibition studies. Because the L2GPeptide was used as the immunogen and was used to screen for positiveclones, it was known that the 5B7 epitope must be within that 19-aminoacid sequence (see SEQ ID NO: 2). The L2G Peptide, a peptide containingthe 13 C-terminal amino acid residues of the L2G Peptide, and a peptidecontaining the six N-terminal amino acid residues of the L2G Peptideplus three additional N-terminal residues (i.e., QIS) corresponding tothe respective positions in the human EGFR sequence. The amino acidsequences of the subject peptides are shown in FIG. 7A. The peptideswere synthesized by Genemed Synthesis, Inc. (South San Francisco,Calif.).

A known EGFR-positive lung squamous cell carcinoma was chosen for thepeptide inhibition study. The 5B7 antibody was pre-incubated with eachpeptide for 1 hour at room temperature before application to the tissue.A 1000-fold molar excess of peptide compared to antibody was used.

As shown in FIG. 7, Peptide 1 resulted in partial inhibition of 5B7binding, which indicated that part of the epitope was contained withinPeptide 1. Peptide 2 resulted in complete inhibition of 5B7 binding,which indicated that the primary epitope was contained in Peptide 2.Logically, the three amino acids shared by Peptides 1 and 2 (i.e., QQD)must contain at least part of the full epitope. Peptide 3 alsocompletely inhibited 5B7 binding; thus, the tyrosine did notsignificantly contribute to the 5B7 epitope. An average epitope is onthe order of 7-12 contiguous amino acids; thus, the boxed residues inFIG. 7 represent a likely full-length 5B7 epitope with some possibilityfor an additional 1-5 residues at the C-terminal end.

Example 5 SOCS3 Knockout Unmasks Clone 5B7 Epitope

The L2G Peptide of the EGFR sequence is within the binding region forSOCS3 (Xia et al., J. Biol. Chem., 277(34):30716-23, 2002). Thus, it waspostulated that SOCS3 may be masking the 5B7 epitope in some tissues. Totest this hypothesis, livers from a hepatic-specific, SOCS3-knockoutmouse were obtained from the laboratory that developed the model (Ogataet al., Gastroenterology, 131(1):179-93, 2006). Sections offormalin-fixed, paraffin-embedded livers from wild-type and SOCS3 micewere stained with clone 5B7 as described in Example 2.

As shown in the left two panels of FIG. 8, 5B7 failed to stain normalliver, which expresses SOCS3. In comparison, as shown in the right twopanels of FIG. 8, 5B7 positively stained the membranes of cells inlivers lacking SOCS3. These results indicate that the loss or absence ofSOCS3 allows for the binding of clone 5B7 to the regulatory domain ofEGFR.

SOCS3 is only one example of a regulatory molecule that directlyinteracts with EGFR. The results demonstrated herein are widelyapplicable to other interface-specific binding molecules that haveepitopes in the interface between two components of a molecular complex,such as between EGFR and its many regulatory proteins.

Example 6 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of NSCLC Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of NSCLC cancer patients toEGFR-inhibitor therapy (IRESSA™)

Tissue arrays containing biopsy samples from at least 100 NSCLC cancerpatients are obtained. Each patient is treated with IRESSA™(EGFR-inhibitor) therapy with a dosage of 250 mg/day given orally. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline above background level of the cell.Cytoplasmic reactivity may be absent or may be whether it is complete ormoderately intense when membrane staining is very intense. incompletecircumferential Submembranous cytoplasmic accentuation may be present.staining in more than 0% 2.5 Intense reactivity: Shades of brownstaining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor cells. 0 No reactivity Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ID-basedtherapy. Patients in categories 1 and 3 will have an objective responseto IRESSA™ therapy and patients in categories 2 and 4 will notsignificantly respond to IRESSA™ therapy.

Example 7 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of NSCLC Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of NSCLC cancer patients toEGFR-inhibitor therapy (TARCEVA™).

Tissue arrays containing biopsy samples from at least 100 NSCLC cancerpatients are obtained. Each patient is treated with TARCEVA™ (EGFRinhibitor) therapy with a dosage of 150 mg/day given orally. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline above background level of the cell.Cytoplasmic reactivity may be absent or may be whether it is complete ormoderately intense when membrane staining is very intense. incompletecircumferential Submembranous cytoplasmic accentuation may be present.staining in more than 0% 2.5 Intense reactivity: Shades of brownstaining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor cells. 0 No reactivity Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ID-basedtherapy. Patients in categories 1 and 3 will have an objective responseto TARCEVA™ therapy and Patients in categories 2 and 4 will notsignificantly respond to TARCEVA™ therapy.

Example 8 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Colorectal Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of colorectal cancer patients toEGFR-inhibitor therapy (ERBITUX™).

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient is treated with ERBITUX™(EGFR inhibitor) therapy with a dosage of 400 mg/m² given i.v. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+ Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick background level whether it isoutline of the cell. Cytoplasmic reactivity may be absent or maycomplete or incomplete be moderately intense when membrane staining isvery intense. circumferential staining in Submembranous cytoplasmicaccentuation may be present. more than 0% tumor cells 2.5 Intensereactivity: Shades of brown staining of medium darkness (intensity).Membranous reactivity is usually but not always complete, producing acircular outline of the neoplastic cell. Incomplete membrane reactivityof moderate intensity is also considered 2+. The cytoplasmic reactivityis of weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor cells. 0 No reactivity Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ED-basedtherapy. Patients in category 1 will have an objective response toERBITUX™ therapy, and patients in categories 2, 3 and 4 will notsignificantly respond to ERBITUX™ therapy.

Example 9 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Colorectal Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of colorectal cancer patients toEGFR-inhibitor therapy (VECTIBIX™).

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient is treated with VECTIBIX™(EGFR inhibitor) therapy with a dosage of 6 mg/kg given i.v. Eachpatient has post-therapy follow-up for up to 5 years. Each biopsy sampleis fixed in 10% NBF and paraffin embedded. Five (5) micron sections ofeach biopsy sample are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline of above background level the cell.Cytoplasmic reactivity may be absent or may be moderately whether it iscomplete or intense when membrane staining is very intense.Submembranous incomplete circumferential cytoplasmic accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately65% of cases are expected to fall into category 1, 19% in category 2,<1% in category 3 and 15% in category 4. Patient outcome is directlyrelated to the scoring category as indicated in Table 2 for an ED-basedtherapy. Patients in category 1 will have an objective response toVECTIBIX™ therapy and, patients in categories 2, 3 and 4 will notsignificantly respond to VECTIBIX™ therapy.

Example 10 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Breast Cancer Patients to EGFR-Inhibitor Therapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of breast cancer patients toEGFR-inhibitor therapy or, more particularly, HER1 (EGFR)/HER2-inhibitortherapy (such as, lapatinib (TYKERB™)).

Tissue arrays containing biopsy samples from at least 100 breast cancerpatients are obtained. Each patient is treated with lapatinib (TYKERB™)(HER1 (EGFR)/HER2-inhibitor) with a dosage of 1250-1500 mg/day givenorally. Each patient has post-therapy follow-up for at least 20 months.Each biopsy sample is fixed in a standard fixative and paraffinembedded. Sections of each biopsy sample (e.g., 5 μm thick) are cut andarrayed on positively charged glass slides. The slides are stained withan RD-binding molecule (e.g., clone 5B7) and an ED-binding molecule(e.g., clone 3C6) according to the protocols in Example 2. The resultingstained array slides are scored by light microscopy by a pathologistaccording to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not always, of tumor cell membranes in a complete membranepattern, producing a thick outline of the cell. above background levelCytoplasmic reactivity may be absent or may be moderately intense whenwhether it is complete or membrane staining is very intense.Submembranous cytoplasmic incomplete circumferential accentuation may bepresent staining in more than 0% 2.5 Intense reactivity: Shades of brownstaining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately20% of cases are expected to fall into category 1, 16% in category 2, 3%in category 3 and 61% in category 4. Patient outcome is directly relatedto the scoring category as indicated in Table 2 for an ID-based therapy.Patients in categories 1 and 3 will have an objective response tolapatinib (TYKERB™) therapy and Patients in categories 2 and 4 will notsignificantly respond to lapatinib (TYKERB™) therapy.

Example 11 EGFR RD-Binding Molecules, Such as Clone 5B7, Predict theResponse of Hepatocellular Carcinoma Cancer Patients to EGFR-InhibitorTherapy

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the response of hepatocellular carcinoma (“HCC”)(such as, resectable HCC) cancer patients to EGFR-inhibitor therapy(IRESSA™)

Tissue arrays containing biopsy samples from at least 100 HCC cancerpatients are obtained (see, for example, samples collected in JS 0414,“A Pilot Study of Adjuvant Therapy of Gefitinib (Iressa, ZD1839) inPatients with Resectable Hepatocellular Carcinoma”, ClinicalTrials.govIdentifier No. NCT00228501). Each patient is treated with IRESSA™(EGFR-inhibitor) therapy with a dosage of 200-500 mg/day given orally.Each patient has post-therapy follow-up for at least 12 months. Eachbiopsy sample is fixed in a standard fixative (e.g., 10% NBF) andparaffin embedded. Sections of each biopsy sample (e.g., 5 nm thick) arecut and arrayed on positively charged glass slides. The slides arestained with an RD-binding molecule (e.g., clone 5B7) and an ED-bindingmolecule (e.g., clone 3C6) according to the protocols in Example 2. Theresulting stained array slides are scored by light microscopy by apathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not always, of tumor cell membranes in a complete membranepattern, producing a thick outline of the cell. above background levelCytoplasmic reactivity may be absent or may be moderately intense whenwhether it is complete or membrane staining is very intense.Submembranous cytoplasmic incomplete circumferential accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 or 3C6). The result of each case isassigned to 1 of the 4 categories described in Table 2. Approximately60% of cases are expected to fall into category 1, 13% in category 2, 5%in category 3 and 22% in category 4. Patient outcome is directly relatedto the scoring category as indicated in Table 2 for an ID-based therapy.Patients in categories 1 and 3 will have an objective response toIRESSA™ therapy and patients in categories 2 and 4 will notsignificantly respond to IRESSA™ therapy.

Example 12 Clone 5B7 Status is a Clear Indicator of Lung CancerPrognosis

This Example demonstrates that clone 5B7 predicts the prognosis of lungcancer patients.

A tissue array containing lung biopsy samples from 109 Stage I or IINSCLC patients was obtained (a subset of the larger cohort described inOlaussen et al., New Engl. J. Med., 355(10):983-991, 2006). None of thepatients from whom the biopsies were obtained had been treated with anEGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™, or TARCEVA™).Patient survival post-diagnosis was monitored on a continuing basis.Each biopsy sample was paraffin embedded, cancerous areas in the biopsywere identified, a core of the cancerous area removed, and placed in adonor array paraffin block. Three to five micron sections of the donorarray block were cut and mounted on glass slides. Slides containingserial sections of the donor array block were stained with clone 5B7 orclone 3C6 according to the protocols in Example 2. The resulting stainedslides are scored by light microscopy by a pathologist according to thefollowing criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+ Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline background level whether itis of the cell. Cytoplasmic reactivity may be absent or may be completeor incomplete moderately intense when membrane staining is very intense.circumferential staining in Submembranous cytoplasmic accentuation maybe present. more than 0% tumor cells 2.5 Intense reactivity: Shades ofbrown staining of medium darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 2+ Moderate reactivity: Shades of brownstaining of intermediate darkness (intensity). Membranous reactivity isusually but not always complete, producing a circular outline of theneoplastic cell. Incomplete membrane reactivity of moderate intensity isalso considered 2+. The cytoplasmic reactivity is of weaker intensitythan the membrane reactivity. 1.5 Slight reactivity: Staining ofintermediate intensity that is membraneous. Cytoplasmic reactivity thatis uniform and involves all the cytoplasm may be present, but should notbe evaluated for positivity. 1+ Weak reactivity: Faint or light brownreactivity that is membranous. Cytoplasmic reactivity that is uniformand involves all the cytoplasm may be present, but should not beevaluated for positivity. Negative: Absence of 0.5 Trace reactivity:Trace brown reactivity where membranous and membrane staining abovecytoplasmic localization is indeterminate. background in all tumorcells. 0 No reactivity Presence of cytoplasmic in the absence ofmembrane staining.

The score for each biopsy sample and the associated follow-up is shownin Table 14.

TABLE 14 Biopsy scores Sample 3C6 5B7 Months at Months at Number ScoreScore Recurrence Death P003 2 2 None None P005 2.5 0.5 35 36 P006 0 2  636 P007 0 2 None None P008 0 1  5 25 P010 3 0.5 None None P011 2.5 0.542 57 P012 2.5 2 None None P014 2 2.5 70 None P015 0 1 None None P016 11 None None P017 2.5 2 17 33 P018 2.5 2 None None P019 3 2 None NoneP020 0.5 1 None None P022 2 1.5 None None P023 0 2 17 None P024 0 0.5None None P025 0 2.5 None None P026 0 2.5 16 94 P029 0.5 1 47 24 P030 02 None None P031 2 1.5 36 70 P033 3 2 None None P037 0.5 4.5 None 96P039 0 1 None None P041 0.5 1.5 None None P042 1 0 None None P044 1 1None None P047 0 2  8 25 P050 1.5 0 None None P051 2.5 0.5 None NoneP057 1 0 90 None P058 0 0.5 None None P061 2 1 None None P063 1.5 3 NoneNone P064 1.5 2.5 65 None P065 0 1.5 None None P066 0.5 0.5 None NoneP068 3 1 None None P070 1 1  5  7 P071 0.5 3 15 18 P072 3 2 22 25 P073 10.5 68 None P074 2.5 0 None None P076 2 0.5 None None P078 3 1.5 NoneNone P080 0.5 3 None None P161 0 0 None None P162 2 2 None None P163 2 1None None P165 1 1 None None P167 0.5 1.5 None None P168 3 3 None NoneP169 2.5 2.5 None None P170 2 2.5 None None P171 1 1 None None P172 00.5 19 28 P173 0 1 None None P174 2 2 None None P175 0.5 2 None NoneP176 0.5 0.5 None None P177 0.5 2 None None P178 2 2 None None P179 32.5 None None P180 1.5 1 None None P181 2.5 2.5 None None P182 3 3 NoneNone P184 3 3 16 44 P187 2 2.5 None None P188 1 1 None None P189 0 1 17None P190 3 3 17 44 P194 1.5 3 None None P197 0 1 None None P200 1 0.5None None P202 3 3  8 20 P203 1.5 2  9 15 P207 1 2.5 None None P209 2.52.5 None None P210 2 1 None None P211 2 3  4  5 P212 1 1.5 None NoneP213 3 3 29 None P214 2 2.5 None None P216 3 3 15 21 P217 3 3 19 NoneP219 2 2 None None P220 2 2 None None P221 0 0.5 None None P222 0 1  612 P223 0 0.5 None None P224 3 2.5 24 53 P225 0 1 None None P226 1.5 2 5  9 P227 0.5 1.5 None None P228 1 2.5 None None P230 1.5 1.5 None NoneP231 1 2 None None P232 0 0.5 None None

As shown in FIG. 9, clone 3C6 staining (whether negative or positive)has no correlation to NSCLC patient overall survivability while clone5B7 clearly delineates two populations. In particular, positive 5B7staining (score=1 or greater) identified NSCLC patients (n=80) havingpoor survivability, and negative 5B7 staining (score <1) identifiedNSCLC patients (n=20) with greater survivability. For example, as shownin FIG. 9B, approximately 82% of patients whose biopsy sample stainednegative for clone 5B7 were still surviving at 8.3 years post-diagnosis.In comparison, approximately 65% of patients whose biopsy sample stainedpositive for clone 5B7 were surviving at the same time point.

As shown in FIG. 10, clone 3C6 staining (whether negative or positive)has no correlation to NSCLC patient disease-free survival (DFS) whileclone 5B7 clearly delineates two populations. In particular, positive5B7 staining (score=1 or greater) identified NSCLC patients (n=80)having poor DFS, and negative 5B7 staining (score <1) identified NSCLCpatients (n=20) with greater DFS. For example, as shown in FIG. 10B,approximately 75% of patients whose biopsy sample stained negative forclone 5B7 were still surviving at 6 years post-diagnosis. In comparison,approximately 62% of patients whose biopsy sample stained positive forclone 5B7 were surviving at the same time point. The 5B7-positive and5B7-negative curves converge around 90 months post-diagnosis most likelydue to a statistical artifact cause by a decrease in the number of5B7-negative samples at that (and later) time points. It is expectedthat 5B7-negative NSCLC patients will continue to have a betterprognosis at 90 months and beyond when an even larger patient cohort isexamined.

This Example demonstrates that EGFR RD-binding molecules, such as clone5B7, predict the prognosis (e.g., overall survival and/or disease-freesurvival) of NSCLC patients (e.g., early stage NSCLC patients)independent of treatment.

Example 13 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Colorectal Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of colorectal cancer patients.

Tissue arrays containing biopsy samples from at least 100 colorectalcancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline of above background level the cell.Cytoplasmic reactivity may be absent or may be moderately whether it iscomplete or intense when membrane staining is very intense.Submembranous incomplete circumferential cytoplasmic accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor cells. 0 No reactivity Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 14 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Head and Neck Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of head and neck cancer patients.

Tissue arrays containing biopsy samples from at least 100 head and neckcancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC 3+ Strong reactivity: Dark brown to black staining is usually,but not always, staining of tumor cell in a complete membrane pattern,producing a thick outline of the cell. membranes above Cytoplasmicreactivity may be absent or may be moderately intense when backgroundlevel whether membrane staining is very intense. Submembranouscytoplasmic it is complete or accentuation may be present. incompletecircumferential 2.5 Intense reactivity: Shades of brown staining ofmedium darkness staining in more than 0% (intensity). Membranousreactivity is usually but not always complete, tumor cells producing acircular outline of the neoplastic cell. Incomplete membrane reactivityof moderate intensity is also considered 2+. The cytoplasmic reactivityis of weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 15 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Gastric Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of gastric cancer patients.

Tissue arrays containing biopsy samples from at least 100 gastric cancerpatients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline of above background level the cell.Cytoplasmic reactivity may be absent or may be moderately whether it iscomplete or intense when membrane staining is very intense.Submembranous incomplete circumferential cytoplasmic accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 16 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Glioblastoma Cancer Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of glioblastoma cancer patients.

Tissue arrays containing biopsy samples from at least 100 glioblastomacancer patients are obtained. Each patient preferably will not have beentreated with an EGFR-based therapy (e.g., ERBITUX™, VECTIBIX™, IRESSA™,or TARCEVA™). Each patient is followed for up to 5 years post-diagnosis.Each biopsy sample is fixed in 10% NBF and paraffin embedded. Five (5)micron sections of each biopsy sample are cut and arrayed on positivelycharged glass slides. The slides are stained with an RD-binding molecule(e.g., clone 5B7) and an ED-binding molecule (e.g., clone 3C6) accordingto the protocols in Example 2. The resulting stained array slides arescored by light microscopy by a pathologist according to the followingcriteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining 3+ Strong reactivity: Dark brown to black staining isusually, but not of tumor cell membranes always, in a complete membranepattern, producing a thick outline of above background level the cell.Cytoplasmic reactivity may be absent or may be moderately whether it iscomplete or intense when membrane staining is very intense.Submembranous incomplete circumferential cytoplasmic accentuation may bepresent. staining in more than 0% 2.5 Intense reactivity: Shades ofbrown staining of medium darkness tumor cells (intensity). Membranousreactivity is usually but not always complete, producing a circularoutline of the neoplastic cell. Incomplete membrane reactivity ofmoderate intensity is also considered 2+. The cytoplasmic reactivity isof weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor 0 No reactivity cells. Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 65% of cases are expected to fall into category 1, 19% incategory 2, <1% in category 3 and 15% in category 4. Patient outcomewill be directly related to the scoring category as indicated in FIG. 3and Table 2. Patients in category 1 and 3 will have a poor prognosis,and patients in categories 2 and 4 will have a better prognosis.

Example 17 EGFR RD-Binding Molecules, Such as Clone 5B7, are Indicatorsof Hepatocellular Carcinoma Prognosis

This Example demonstrates that a disclosed RD-binding molecule (e.g.,clone 5B7) predicts the prognosis of HCC (such as, resectable HCC)cancer patients

Tissue arrays containing biopsy samples from at least 100 HCC cancerpatients are obtained (see, for example, control arm of samplescollected in JS 0414, “A Pilot Study of Adjuvant Therapy of Gefitinib(Iressa, ZD1839) in Patients with Resectable Hepatocellular Carcinoma”,ClinicalTrials.gov Identifier No. NCT00228501). Each patient preferablywill not have been treated with an EGFR-based therapy (e.g., ERBITUX™,VECTIBIX™, IRESSA™, or TARCEVA™). Each patient is followed for up to 5years post-diagnosis. Each biopsy sample is fixed in a standard fixative(e.g., 10% NBF) and paraffin embedded. Sections of each biopsy sample(e.g., 5 μm thick) are cut and arrayed on positively charged glassslides. The slides are stained with an RD-binding molecule (e.g., clone5B7) and an ED-binding molecule (e.g., clone 3C6) according to theprotocols in Example 2. The resulting stained array slides are scored bylight microscopy by a pathologist according to the following criteria:

Staining Intensity Report Result Score Microscope Observation Positive:Any IHC staining of 3+ Strong reactivity: Dark brown to black stainingis usually, but not tumor cell membranes above always, in a completemembrane pattern, producing a thick outline of background level whetherit is the cell. Cytoplasmic reactivity may be absent or may bemoderately complete or incomplete intense when membrane staining is veryintense. Submembranous circumferential staining in cytoplasmicaccentuation may be present. more than 0% tumor cells 2.5 Intensereactivity: Shades of brown staining of medium darkness (intensity).Membranous reactivity is usually but not always complete, producing acircular outline of the neoplastic cell. Incomplete membrane reactivityof moderate intensity is also considered 2+. The cytoplasmic reactivityis of weaker intensity than the membrane reactivity. 2+ Moderatereactivity: Shades of brown staining of intermediate darkness(intensity). Membranous reactivity is usually but not always complete,producing a circular outline of the neoplastic cell. Incomplete membranereactivity of moderate intensity is also considered 2+. The cytoplasmicreactivity is of weaker intensity than the membrane reactivity. 1.5Slight reactivity: Staining of intermediate intensity that ismembraneous. Cytoplasmic reactivity that is uniform and involves all thecytoplasm may be present, but should not be evaluated for positivity. 1+Weak reactivity: Faint or light brown reactivity that is membranous.Cytoplasmic reactivity that is uniform and involves all the cytoplasmmay be present, but should not be evaluated for positivity. Negative:Absence of 0.5 Trace reactivity: Trace brown reactivity where membranousand membrane staining above cytoplasmic localization is indeterminate.background in all tumor cells. 0 No reactivity Presence of cytoplasmicin the absence of membrane staining.

The score for each case is recorded in a database comparing the scorefor each binding molecule (e.g., 5B7 and 3C6). The results of each casewill fall into one of the 4 categories described in Table 2.Approximately 60% of cases are expected to fall into category 1, 13% incategory 2, 5% in category 3 and 22% in category 4. Patient outcome willbe directly related to the scoring category as indicated in FIG. 3 andTable 2. Patients in category 1 and 3 will have a poor prognosis, andpatients in categories 2 and 4 will have a better prognosis.

While this disclosure has been described with an emphasis uponparticular embodiments, it will be obvious to those of ordinary skill inthe art that variations of the particular embodiments may be used and itis intended that the disclosure may be practiced otherwise than asspecifically described herein. Accordingly, this disclosure includes allmodifications encompassed within the spirit and scope of the disclosureas defined by the following claims:

1. A method of identifying a diagnostic reagent, comprising: identifyinga receptor tyrosine kinase (RTK), which is a component of a cellularpathway that confers a growth advantage on a cancer cell, whereininhibition of the RTK decreases the cancer cell growth advantage andwherein a function of the RTK protein is positively or negativelyregulated by a regulatory protein when the RTK and the regulatoryprotein form a protein-protein interaction, thereby producing aprotein-protein interface; and producing a specific binding reagent thatspecifically binds to at least one RTK amino acid of the protein-proteininterface in the absence of the protein-protein interaction and fails tospecifically bind the at least one RTK amino acid of the protein-proteininterface in the presence of the protein-protein interaction, whereinthe specific binding reagent is a diagnostic reagent.
 2. The method ofclaim 1, further comprising identifying five or more amino acid residuesthat form the protein-protein interface, and wherein the specificbinding reagent specifically binds to the five or more amino acidresidues that form the protein-protein interface in the absence of theprotein-protein interaction and fails to specifically bind the five ormore amino acid residues that form the protein-protein interface in thepresence of the protein-protein interaction.
 3. The method of claim 1,wherein the RTK protein comprises an RTK listed in Table
 2. 4. Themethod of claim 1, wherein a function of the RTK protein is negativelyregulated by a regulatory protein when the RTK and the regulatoryprotein form a protein-protein interaction, and wherein the negativeregulation decreases the cancer cell growth advantage.
 5. The method ofclaim 1, wherein a function of the RTK protein is positively regulatedby a regulatory protein when the RTK and the regulatory protein form aprotein-protein interaction, and wherein the positive regulationincreases the cancer cell growth advantage.
 6. The method of claim 1,wherein the specific binding reagent comprises an antibody or fragmentthereof, or an aptamer.
 7. The method of claim 1, wherein identifyingthe five or more amino acid residues comprises competitively bindingpeptides and the regulatory protein to the RTK, wherein the peptidecomprises at least 5 amino acid residues of the protein-proteininterface.
 8. The method of claim 1, wherein the protein-proteininterface comprises an RTK intracellular domain.
 9. The method of claim8, wherein the specific binding reagent specifically binds to at least 5amino acids of the RTK intracellular domain that form theprotein-protein interface.
 10. The method of claim 1, wherein producinga specific binding reagent comprises: identifying peptide sequences ofless than about 20 amino acids that form the protein-protein interfaceand that have significant antigenicity; and administering at least oneof the peptide sequences to an animal under conditions that permitproduction of the specific binding reagent in the animal.
 11. The methodof claim 1, wherein the specific binding reagent does not specificallybind to an RTK extracellular domain, an RTK transmembrane domain, or theregulatory protein.
 12. A diagnostic reagent identified using the methodof claim
 1. 13. A kit comprising: the diagnostic reagent of claim 12 ina container; and a labeled secondary antibody.
 14. A method ofdiagnosing a subject having cancer, comprising: contacting a sampleobtained from the subject with the diagnostic reagent of claim 12,wherein the diagnostic reagent specifically binds the one or more aminoacid residues in the absence of the protein-protein interaction betweenthe RTK protein and a negative regulatory protein; and detecting bindingof the diagnostic reagent with a protein in the sample, wherein thepresence of detectable binding indicates that the RTK protein did notform a protein-protein interaction with the negative regulatory proteinand the subject is diagnosed to have a cancer with worse prognosis andabsence of detectable binding indicates that the RTK protein formed aprotein-protein interaction with the negative regulatory protein and thesubject is diagnosed to have cancer with a good prognosis.
 15. Themethod of claim 14, wherein the method is a method of prognosing asensitivity of the cancer to an RTK inhibitor, wherein the presence ofdetectable binding indicates that the cancer will be sensitive to theRTK inhibitor and absence of detectable binding indicates that thecancer will be resistant to the RTK inhibitor.
 16. The method of claim15, wherein the RTK inhibitor comprises a tyrosine kinase inhibitor(TKI).
 17. The method of claim 14, further comprising: contacting thesample with an agent that permits detection of an RTK extracellulardomain, wherein the presence of a detectable signal from the agentindicates that an RTK extracellular domain is present in the sample, andthe absence of a detectable signal from the agent indicates that the RTKextracellular domain is absent or mutated.
 18. The method of claim 17,wherein the method is a method of prognosing a sensitivity of the cancerto an RTK extracellular domain-based inhibitor, wherein the presence ofdetectable binding by the diagnostic reagent that specifically binds theone or more amino acid residues in the absence of the protein-proteininteraction between the RTK protein and a negative regulatory proteinand the presence of detectable binding by the agent that permitsdetection of an RTK extracellular domain indicates that the cancer willbe sensitive to RTK inhibitors that target the RTK extracellular orintracellular domain; wherein the absence of detectable binding by thediagnostic reagent that specifically binds the one or more amino acidresidues in the absence of the protein-protein interaction between theRTK protein and a negative regulatory protein and the presence ofdetectable binding by the agent that permits detection of an RTKextracellular domain indicates that the cancer will be resistant to RTKinhibitors that target the RTK extracellular or intracellular domain;wherein the presence of detectable binding by the diagnostic reagentthat specifically binds the one or more amino acid residues in theabsence of the protein-protein interaction between the RTK protein and anegative regulatory protein and the absence of detectable binding by theagent that permits detection of an RTK extracellular domain indicatesthat the cancer will be resistant to RTK extracellular domain-basedinhibitors but sensitive to RTK inhibitors that target the RTKintracellular domain; and wherein the absence of detectable binding bythe diagnostic reagent that specifically binds the one or more aminoacid residues in the absence of the protein-protein interaction betweenthe RTK protein and a negative regulatory protein and the absence ofdetectable binding by the agent that permits detection of an RTKextracellular domain indicates that the cancer will not be responsive toRTK inhibitors that target the RTK extracellular or intracellulardomain.
 19. The method of claim 18, wherein the RTK extracellulardomain-based inhibitor comprises an antibody specific for the RTKextracellular domain.
 20. A method of diagnosing a subject, comprising:contacting a sample obtained from the subject with the diagnosticreagent of claim 12, wherein the diagnostic reagent specifically bindsthe one or more amino acid residues in the absence of theprotein-protein interaction between the RTK protein and a positiveregulatory protein; and detecting binding of the diagnostic reagent witha protein in the sample, wherein the presence of detectable bindingindicates that the RTK protein did not form a protein-proteininteraction with the positive regulatory protein and the subject isdiagnosed to have a cancer with good prognosis and absence of detectablebinding indicates that the RTK protein formed a protein-proteininteraction with the positive regulatory protein and the subject isdiagnosed to have cancer with a poor prognosis.
 21. The method of claim14, wherein the sample comprises a formalin-fixed, paraffin-embeddedtissue sample.